Method for producing α-L-aspartyl-L-phenylalanine-β-ester and method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester

ABSTRACT

A method for producing an α-L-aspartyl-L-phenylalanine-β-ester (also named α-L-(β-o-substituted aspartyl)-L-phenylalanine), which is an intermediate of an α-L-aspartyl-L-phenylalanine-α-methyl ester (also named α-L-aspartyl-L-phenylalanine methyl ester; product name: aspartame), easily, at high yield and inexpensively without going through a complex synthesis method is provided. Further, an easy, inexpensive and high-yield production method for an α-L-aspartyl-L-phenylalanine-α-methyl ester is provided. α-L-aspartyl-L-phenylalanine-α-methyl ester is produced from a L-aspartic acid-α,β-diester and L-phenylalanine using an enzyme or enzyme-containing substance that has an ability to catalyze a reaction in which L-phenylalanine performs no nucleophilic attack on a β-ester site of L-aspartic acid-α,β-diester but performs a nucleophilic attack on an α-ester site thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of PCT/JP2004/000620 filed onJan. 23, 2004, which claims priority to JP 2003-016764, filed on Jan.24, 2003, JP 2003-201819, filed on Jul. 25, 2003, and U.S. 60/491,546,filed Aug. 1, 2003.

TECHNICAL FIELD

The present invention relates to a method for producing anα-L-aspartyl-L-phenylalanine-β-ester (also named as“α-L-(β-o-substituted aspartyl)-L-phenylalanine (abbreviation: α-ARP))and to a method for producing an α-L-aspartyl-L-phenylalanine-α-methylester(also named α-L-aspartyl-L-phenylalanine methyl ester(abbreviation: α-APM). More particularly, the present invention relatesto a method for producing an α-L-aspartyl-L-phenylalanine-β-ester, whichis an important intermediate for producing anα-L-aspartyl-L-phenylalanine-α-methyl ester (product name: aspartame)that is in great demand as a sweetener, and to a method for producing anα-L-aspartyl-L-phenylalanine-α-methyl ester utilizing the method forproducing the α-L-aspartyl-L-phenylalanine-β-ester.

BACKGROUND ART

Conventionally known methods for producingα-L-aspartyl-L-phenylalanine-α-methyl ester (hereinafter, “α-APM” forshort in some cases) include a chemical synthesis method and enzymaticsynthesis method. As the chemical synthesis method, there has been knowna method for condensing an N-protected L-aspartic acid anhydride withL-phenylalanine methyl ester to synthesize an N-protected APM andeliminating the N-protecting group to obtain APM, and as the enzymaticsynthesis method, there has been known a method for condensing anN-protected L-aspartic acid with L-phenylalanine methyl ester tosynthesize an N-protected APM and eliminating the N-protecting group toobtain APM. In both of the methods, however, steps of introducing aprotecting group and eliminating the protecting group are necessary andthe processes are very troublesome. On the other hand, an APM productionmethod in which no N-protecting group is used has been studied (seeJapanese Patent Publication No. H02-015196 Gazette). However, thismethod is not suitable for industrial production due to very low yieldof the product. Thus, under such circumstances, development ofindustrial production methods for aspartame at lower cost has beendesired.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide a method forproducing an α-L-aspartyl-L-phenylalanine-β-ester, which is anintermediate of an α-L-aspartyl-L-phenylalanine-α-methyl ester, easily,inexpensively and at high yield without going through a complexsynthesis method. Further, it is an object of the present invention toprovide a method for producing an α-L-aspartyl-L-phenylalanine-α-methylester easily, inexpensively, and at high yield.

As a result of conducting extensive research in consideration of theabove objects, the inventors of the present invention have found that anewly discovered enzyme or an enzyme-containing substance is capable ofselectively producing an α-L-aspartyl-L-phenylalanine-β-ester from anL-aspartic acid-α,β-diester and L-phenylalanine, and have achieved thepresent invention.

Namely, the present invention is as described below.

-   [1] A method of producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine), comprising    forming the α-L-aspartyl-L-phenylalanine-β-ester from L-aspartic    acid-α,β-diester and L-phenylalanine using an enzyme or    enzyme-containing substance that has an ability to selectively link    L-phenylalanine to an α-ester site of the L-aspartic    acid-α,β-diester through a peptide bond.-   [2] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [1] above, wherein the enzyme or enzyme-containing substance is one    type or two or more types selected from the group consisting of a    culture of a microbe that has an ability to selectively link    L-phenylalanine to an α-ester site of the L-aspartic    acid-α,β-diester through a peptide bond, a microbial cell separated    from the culture and a treated microbial cell product of the    microbe.-   [3] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2] above, wherein the microbe is a microbe belonging to a genus    selected from the group consisting of Aeromonas, Azotobacter,    Alcaligenes, Brevibacterium, Corynebacterium, Escherichia,    Empedobacter, Flavobacterium, Microbacterium, Propionibacterium,    Brevibacillus, Paenibacillus, Pseudomonas, Serratia,    Stenotrophomonas, Sphingobacterium, Streptomyces, Xanthomonas,    Williopsis, Candida, Geotrichum, Pichia, Saccharomyces, Torulaspora,    Cellulophaga, Weeksella, Pedobacter, Persicobacter, Flexithrix,    Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens,    Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobacillus,    Tenacibaculum, Rhodotermus, Zobellia, Muricauda, Salegentibacter,    Taxeobacter, Cytophaga, Marinilabilia, Lewinella, Saprospira, and    Haliscomenobacter.-   [4] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2] above, wherein the microbe is a transformed microbe that is    capable of expressing a protein (A) or (B):    -   (A) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 616 of an amino acid sequence        described in SEQ ID NO: 6 of the Sequence Listing,    -   (B) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 23 to 616 of the amino acid        sequence described in SEQ ID NO: 6 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [5] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2], wherein the microbe is a transformed microbe that is capable of    expressing a protein (C) or (D):    -   (C) a protein having an amino acid sequence consisting of amino        acid residues numbers 21 to 619 of an amino acid sequence        described in SEQ ID NO: 12 of the Sequence Listing,    -   (D) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 21 to 619 of the amino acid        sequence described in SEQ ID NO: 12 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [6] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2], wherein the microbe is a transformed microbe that is capable of    expressing a protein (E) or (F) below:    -   (E) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 625 of an amino acid sequence        described in SEQ ID NO: 18 of the Sequence Listing,    -   (F) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 23 to 625 of the amino acid        sequence described in SEQ ID NO: 18 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [7] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2] above, wherein the microbe is a transformed microbe that is    capable of expressing a protein (G) or (H) below:    -   (G) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 645 of an amino acid sequence        described in SEQ ID NO: 23 of the Sequence Listing,    -   (H) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 23 to 645 of the amino acid        sequence described in SEQ ID NO: 23 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [8] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2], wherein the microbe is a transformed microbe that is capable of    expressing a protein (I) or (J) below:    -   (I) a protein having an amino acid sequence consisting of amino        acid residues numbers 26 to 620 of an amino acid sequence        described in SEQ ID NO: 25 of the Sequence Listing,    -   (J) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 26 to 620 of the amino acid        sequence described in SEQ ID NO: 25 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [9] The method for producing an α-L-aspartyl-L-phenylalanine-β-ester    (i.e., α-L-(β-o-substituted aspartyl)-L-phenylalanine) according to    [2] above, wherein the microbe is a transformed microbe that is    capable of expressing a protein (K) or (L) below:    -   (K) a protein having an amino acid sequence consisting of amino        acid residues numbers 18 to 644 of an amino acid sequence        described in SEQ ID NO: 27 of the Sequence Listing,    -   (L) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues 18 to 644 of the amino acid        sequence described in SEQ ID NO: 27 of the Sequence Listing, and        having activity to selectively link L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond.-   [10] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [2], wherein the microbe is    a transformed microbe that is capable of expressing a protein (M)    or (N) below:    -   (M) a protein having an amino acid sequence described in SEQ ID        NO: 6 of the Sequence Listing,    -   (N) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 6 of the        Sequence Listing, and having activity to selectively link        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond.-   [11] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [2], wherein the microbe is    a transformed microbe that is capable of expressing a protein (O)    or (P) below:    -   (O) a protein having an amino acid sequence described in SEQ ID        NO: 12 of the Sequence Listing,    -   (P) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 12 of the        Sequence Listing, and having activity to selectively link        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond.-   [12] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to claim 2, wherein the microbe    is a transformed microbe that is capable of expressing a protein (Q)    or (R) below:    -   (Q) a protein containing a mature protein region, having an        amino acid sequence described in SEQ ID NO: 18 of the Sequence        Listing,    -   (R) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        described in SEQ ID NO: 18 of the Sequence Listing, and having        activity to selectively link L-phenylalanine to an α-ester site        of the L-aspartic acid-α,β-diester through a peptide bond.-   [13] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [3], wherein the microbe is    a transformed microbe that is capable of expressing a protein (S)    or (T) below:    -   (S) a protein having an amino acid sequence described in SEQ ID        NO: 23 of the Sequence Listing,    -   (T) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 23 of the        Sequence Listing, and having activity to selectively link        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond.-   [14] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [2], wherein the microbe is    a transformed microbe that is capable of expressing a protein (U)    or (V) below:    -   (U) a protein having an amino acid sequence described in SEQ ID        NO: 25 of the Sequence Listing,    -   (V) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 25 of the        Sequence Listing, and having activity to selectively link        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond.-   [15] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [2] above, wherein the    microbe is a transformed microbe that is capable of expressing a    protein (W) or (X) below:    -   (W) a protein having an amino acid sequence described in SEQ ID        NO: 27 of the Sequence Listing,    -   (X) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 27 of the        Sequence Listing, and having activity to selectively link        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond.-   [16] The method for producing an    α-L-aspartyl-L-phenylalanine-β-ester (i.e., α-L-(β-o-substituted    aspartyl)-L-phenylalanine) according to [1] above, wherein the    enzyme is at least one selected from the group consisting (A) to (X)    below:    -   (A) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 616 of an amino acid sequence        described in SEQ ID NO: 6 of the Sequence Listing,    -   (B) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 616 of the amino        acid sequence described in SEQ ID NO: 6 of the Sequence Listing,        and having activity to selectively linking L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond,    -   (C) a protein having the amino acid sequence consisting of amino        acid residue numbers 21 to 619 of an amino acid sequence        described in SEQ ID NO: 12 of the Sequence Listing,    -   (D) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residue numbers 21 to 619 of the amino        acid sequence described in SEQ ID NO: 12 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (E) a protein having the amino acid sequence consisting of amino        acid residues numbers 23 to 625 of an amino acid sequence        described in SEQ ID NO: 18 of the Sequence Listing,    -   (F) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 625 of the amino        acid sequence described in SEQ ID NO: 18 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (G) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 645 of an amino acid sequence        described in SEQ ID NO: 23 of the Sequence Listing,    -   (H) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 645 of the amino        acid sequence described in SEQ ID NO: 23 of the Sequence        Listing, and activity to selectively linking L-phenylalanine to        an α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond,    -   (I) a protein having an amino acid sequence consisting of amino        acid residues numbers 26 to 620 of an amino acid sequence        described in SEQ ID NO: 25 of the Sequence Listing,    -   (J) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 26 to 620 of the amino        acid sequence described in SEQ ID NO: 25 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (K) a protein having an amino acid sequence consisting of amino        acid residues numbers 18 to 644 of an amino acid sequence        described in SEQ ID NO: 27 of the Sequence Listing,    -   (L) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 18 to 644 of the amino        acid sequence described in SEQ ID NO: 27 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (M) a protein having an amino acid sequence described in SEQ ID        NO: 6 of the Sequence Listing,    -   (N) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 6 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (O) a protein having the amino acid sequence described in SEQ ID        NO: 12 of the Sequence Listing,    -   (P) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 12 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (Q) a protein having an amino acid sequence described in SEQ ID        NO: 18 of the Sequence Listing,    -   (R) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 18 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (S) a protein having an amino acid sequence described in SEQ ID        NO: 23 of the Sequence Listing,    -   (T) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 23 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (U) a protein having an amino acid sequence described in SEQ ID        NO: 25 of the Sequence Listing,    -   (V) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 25 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (W) a protein having an amino acid sequence described in SEQ ID        NO: 27 of the Sequence Listing, and    -   (X) a protein containing a mature protein region, having an        amino acid sequence in the amino acid sequence described in SEQ        ID NO: 27 of the Sequence Listing, and having activity to        selectively linking L-phenylalanine to an α-ester site of the        L-aspartic acid-α,β-diester through a peptide bond.-   [17] The method of producing an    α-L-aspartyl-L-phenylalanine-α-methyl ester (i.e.,    α-L-aspartyl-L-phenylalanine methyl ester), comprising: a reaction    step of synthesizing an α-L-aspartyl-L-phenylalanine-β-methyl ester    (also named α-L-(β-o-methyl aspartyl)-L-phenylalanine (abbreviation:    α-AMP)) by a method of producing an    α-L-aspartyl-L-phenylalanine-β-ester according to any one of claims    1 to 16; and a reaction step of converting the    α-L-aspartyl-L-phenylalanine-β-methyl ester (i.e., α-L-(β-o-methyl    aspartyl)-L-phenylalanine) to α-L-aspartyl-L-phenylalanine-α-methyl    ester.

By the present invention, α-L-aspartyl-L-phenylalanine-β-ester can beeasily produced. By the method of the present invention,α-L-aspartyl-L-phenylalanine-β-ester can be produced easily and at highyield with reduced use of complicated synthetic methods such asintroduction/elimination of protecting groups.

Furthermore, by the present invention,α-L-aspartyl-L-phenylalanine-α-methyl ester can be produced easily, athigh yield, and inexpensively.

The other objects, features and advantages of the present invention arespecifically set forth in or will become apparent from the followingdetailed descriptions of the invention when read in conjunction with theaccompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram showing amounts of enzymes that exist in a cytoplasmfraction (Cy) and a periplasm fraction (Pe).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in the order of

-   <1> Method of producing α-L-aspartyl-L-phenylalanine-β-ester-   1. Method of producing α-L-aspartyl-L-phenylalanine-β-ester-   2. Microbes used in the present invention-   3. Enzymes used in the present invention; and-   <2> Method of producing α-L-aspartyl-L-phenylalanine-α-methyl ester.-   <1> Method of Producing α-L-aspartyl-L-phenylalanine-β-ester    1. Method of Producing α-L-aspartyl-L-phenylalanine-β-ester

In the method of producing α-L-aspartyl-L-phenylalanine-β-ester of thepresent invention (hereinafter also called “the production method of apeptide of the present invention”), L-phenylalanine and L-asparticacid-α,β-diester are allowed to react in the presence of an enzymehaving a stated peptide forming activity. That is, in the productionmethod of a peptide of the present invention, anα-L-aspartyl-L-phenylalanine-β-ester is formed from an L-asparticacid-α,β-diester and L-phenylalanine using an enzyme orenzyme-containing substance capable of selectively linkingL-phenylalanine to the α-ester site of L-aspartic acid-α,β-diesterthrough a peptide bond. The enzyme or enzyme-containing substancecapable of selectively linking L-phenylalanine to the α-ester site of aL-aspartic acid-α,β-diester refers to an enzyme or enzyme-containingsubstance having an ability or activity to catalyze a reaction in whichsubstantially, L-phenylalanine is able to perform no nucleophilic attackto a β-ester site of L-aspartic acid-α,β-diester but performs anucleophilic attack on an α-ester site thereof only. As shown in thereference example hereinbelow, however, an enzyme or enzyme-containingsubstance has also been obtained that has an ability to catalyze areaction in which substantially, L-phenylalanine is able to perform noattack on the α-ester site of an L-aspartic acid-α,β-diester butperforms nucleophilic attack on the β-ester site thereof only, contraryto the above-mentioned ability, and that produces aβ-L-aspartyl-L-phenylalanine-α-ester (also named β-L-(α-o-substitutedaspartyl)-L-phenylalanine (abbreviation: β-ARP) from the L-asparticacid-α,β-diester and L-phenylalanine.

The reaction formula in which L-phenylalanine performs nucleophilicattack on the α-ester site of L-aspartic acid-α,β-diester to produce anα-L-aspartyl-L-phenylalanine-β-ester (α-ARP) is shown in the followingformula (I-α) (wherein “Me” represents a methyl group) by citing thecase where L-aspartic acid-α,β-dimethyl ester is used as the L-asparticacid-α,β-diester. As shown in the formula (I-α), in the peptideproduction method of the present invention, the amino group ofL-phenylalanine reacts with the α-methyl ester site of the L-asparticacid-α,β-dimethyl ester to form a peptide bond. On the other hand, thefollowing formula (I-β) indicates a reaction in which the β-methyl estersite of L-aspartic acid-α,β-dimethyl ester undergoes nucleophilic attackto form β-L-aspartyl-L-phenylalanine-α-methyl ester (also namedβ-L-(α-o-methyl aspartyl)-L-pphenylalanine (abbreviation: β-AMP)). Thepeptide bond in β-AMP is formed at the β-methyl ester site of theL-aspartic acid-α,β-dimethyl ester. The enzyme or enzyme-containingsubstance used in the present invention accelerates substantially only areaction as in the formula (I-α) but causes substantially no reaction asin the formula (I-β). α-APM can be produced from α-AMP through a simplereaction step (formula (II)), but α-APM cannot be produced directly fromβ-AMP. That is, the method of the present invention is extremelyefficient as a method for producing an intermediate of α-APM and isuseful for industrial production.

A method for allowing the enzyme or enzyme-containing substance to acton L-aspartic acid-α,β-diester and L-phenylalanine may be performed bymixing the enzyme or enzyme-containing substance with L-asparticacid-α,β-diester and L-phenylalanine. More specifically, there may beused a method in which the enzyme or enzyme-containing substance isadded to a solution containing an L-aspartic acid-diester andL-phenylalanine to effect reaction. When a microbe which produces theenzyme is used as the enzyme-containing substance, either the reactionmay be carried out as described above, or a method which includesculturing a microbe that produces the enzyme to produce and accumulatethe enzyme in the microbe or a culture liquid in which the microbe hasbeen cultured, and adding an L-aspartic acid-α,β-diester andL-phenylalanine to the culture liquid, or the like method may be used.The thus produced α-L-aspartyl-L-phenylalanine-β-ester is recoveredaccording to the conventional method and it can be purified, ifnecessary.

The “enzyme-containing substance” may be any substance so far as itcontains the enzyme, and specific modes thereof include a culture of amicrobe which produces the enzyme, a microbial cell separated from theculture and a treated microbial cell product of the microbe. The cultureof microbe means a substance obtained by culturing a microbe, andspecifically means a mixture of microbial cell, a medium used forculturing the microbe and a substance produced by the cultured microbe,and so forth. In addition, the microbial cell may be washed to use as awashed microbial cell. Moreover, the treated microbial cell productincludes those obtained by subjecting the microbial cell to crushing,lysis, or freeze-drying, and further a crude enzyme recovered bytreating the microbial cell and a purified enzyme obtained by furtherpurification. As the purification-treated enzyme, a partially purifiedenzyme obtained by various purification methods and so forth may beused. In addition, immobilized enzymes which have been immobilized by acovalent bonding method, an adsorption method, an entrapment method, orthe like may be used. Further, for some microbes to be used, a portionof the microbial cells may undergo lysis during culturing and in such acase, the supernatant of the culture liquid may be utilized as theenzyme-containing substance as well.

In addition, as the microbe that contains the enzyme, a wild strain maybe used or a gene recombinant strain in which the enzyme has beenexpressed may be used. Such microbe is not limited to an enzymemicrobial cell but the treated microbial cell products such asacetone-treated microbial cell and freeze-dried microbial cell may beused. Further, immobilized microbial cells obtained by immobilizing thetreated microbial cell product using a covalent bonding method, anadsorption method, an entrapment method, or the like, or an immobilizedtreated microbial cell product may be used.

Use of a wild strain which is able to produce a peptide forming enzymehaving an activity to form an α-L-aspartyl-L-phenylalanine-β-ester ispreferred in that peptide production can be performed more readilywithout going through a step of making a gene recombinant strain. On theother hand, a gene recombinant strain which has been transformed so asto express a peptide forming enzyme having an activity to produce anα-L-aspartyl-L-phenylalanine-β-ester can be modified such that thepeptide forming enzyme is produced in a larger amount. Thus, it ispossible to synthesize an α-L-aspartyl-L-phenylalanine-β-ester in alarger amount and at a higher rate. Culturing a microbe of wild strainor gene recombinant strain in a medium to accumulate the peptide formingenzyme in the medium and/or microbe, and mixing the thus accumulatedproduct with an L-aspartic acid-α,β-diester and L-phenylalanine can forman α-L-aspartyl-L-phenylalanine-β-ester.

Note that when cultured products, cultured microbial cells, washedmicrobial cells and treated microbial cell products obtained bysubjecting microbial cells to crushing or lysis are used, it is oftenthe case that an enzyme exists that decomposes the formedα-L-aspartyl-L-phenylalanine-β-ester without being involved in theformation of the α-L-aspartyl-L-phenylalanine-β-ester. In such a case,it is preferred in some occasions to add a metal protease inhibitor suchas ethylenediaminetetraacetic acid (EDTA). The addition amount is in therange of 0.1 millimolar (mM) to 300 mM, preferably 1 mM to 100 mM.

The amount of enzyme or enzyme-containing substance used may be enoughif it is an amount at which the target effect is demonstrated (effectiveamount). While a person with ordinary skill in the art can easilydetermine this effective amount through simple, preliminaryexperimentation, the use amount is, for example, about 0.01 to about 100units (“U”) in the case of using enzyme, and about 0.1 to about 500 g/Lin the case of using washed microbial cells. Note that 1 U is defined tobe an amount of enzyme which allows production of 1 micromole (μmole) ofL-α-aspartyl-L-phenylalanine-β-methyl ester from 100 mM L-asparticacid-α,β-dimethyl ester and 200 mM L-phenylalanine at 25° C. in oneminute.

The L-aspartic acid-α,β-diester to be used in the reaction may be anyone that is condensed with L-phenylalanine to produce anα-L-aspartyl-L-phenylalanine-β ester. Examples of the L-asparticacid-α,β-diester include L-aspartic acid-α,β-dimethyl ester andL-aspartic acid-α,β-diethyl ester. When L-aspartic acid-α,β-dimethylester and L-phenylalanine are allowed to react,α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) is produced, andwhen L-aspartic acid-α,β-diethyl ester and L-phenylalanine are reacted,α-L-aspartyl-L-phenylalanine-β-ethyl ester (also named α-L-(β-o-ethylaspartyl)-L-phenylalanine (abbreviation: α-AEP)) is produced.

While the concentrations of L-aspartic acid-α,β-diester andL-phenylalanine serving as starting materials are each 1 mM to 10 mM,and preferably 0.05 M to 2 M, there may be cases in which it ispreferable to add either one of the substrates in an equimolar amount ormore with respect to the other substrate, and selection is made asnecessary. In addition, in cases where high concentrations of substratesinhibit the reaction, these can be adjusted to concentrations that donot cause inhibition and successively added during the reaction.

The reaction temperature that allows production ofα-L-aspartyl-L-phenylalanine-β-ester is 0 to 60° C., and preferably 5 to40° C. In addition, the reaction pH that allows production ofα-L-aspartyl-L-phenylalanine-β-ester is 6.5 to 10.5, and preferably 7.0to 10.0.

2. Microbes Used in the Present Invention.

As the microbes to be used in the present invention, those microbeswhich have an ability to produce α-L-aspartyl-L-phenylalanine-β-esterfrom an L-aspartic acid-α,β-diester and L-phenylalanine may be usedwithout particular limitation. The microbes that have an ability toproduce α-L-aspartyl-L-phenylalanine-β-ester from an L-asparticacid-α,β-diester and L-phenylalanine include, for example, microbesbelonging to the genera Aeromonas, Azotobacter, Alcaligenes,Brevibacterium, Corynebacterium, Escherichia, Empedobacter,Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus,Paenibacillus, Pseudomonas, Serratia, Stenotrophomonas,Sphingobacterium, Streptomyces, Xanthomonas, Williopsis, Candida,Geotrichum, Pichia, Saccharomyces, Torulaspora, Cellulophaga, Weeksella,Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium,Runella, Thermonema, Psychroserpens, Gelidibacter, Dyadobacter,Flammeovirga, Spirosoma, Flectobacillus, Tenacibaculum, Rhodotermus,Zobellia, Muricauda, Salegentibacter, Taxeobacter, Cytophaga,Marinilabilia, Lewinella, Saprospira, and Haliscomenobacter.

Specifically, the following may be exemplified.

-   -   Aeromonas hydrophila ATCC 13136    -   Azotobacter vinelandii IFO 3741    -   Alcaligenes faecalis FERM P-8460    -   Brevibacterium minutiferuna FERM BP-8277    -   Corynebacterium flavescens ATCC 10340    -   Escherichia coli FERM BP-8276    -   Empedobacter brevis ATCC 14234    -   Flavobacterium resinovorum ATCC 14231    -   Microbacterium arborescens ATCC 4348    -   Propionibacterium shermanii FERM BP-8100    -   Brevibacillus parabrevis ATCC 8185    -   Paenibacillus alvei IFO 14175    -   Pseudomonas fragi IFO 3458    -   Serratia grimesii ATCC 14460    -   Stenotrophomonas maltophilia ATCC 13270    -   Sphingobacterium sp. FERM BP-8124    -   Streptomyces griseolus NRRL B-1305        -   (Streptomyces lavendulae)    -   Xanthomonas maltophilia FERM BP-5568    -   Williopsis saturnus IFO 0895    -   Candida magnoliae IFO 0705    -   Geotrichum fragrance CBS 152.25        -   (Geotrichum amycelium)    -   Geotrichum amycelium IFO 0905    -   Pichia ciferrii IFO 0905    -   Saccharomyces unisporus IFO 0724    -   Torulaspora delbrueckii IFO 0422    -   Cellulophaga lytica NBRC 14961    -   Weeksella virosa NBRC 16016    -   Pedobacter heparinus NBRC 12017    -   Persicobacter diffluens NBRC 15940    -   Flexithrix dorotheae NBRC 15987    -   Chitinophaga pinensis NBRC 15968    -   Cyclobacterium marinum ATCC 25205    -   Runella slithyformis ATCC 29530    -   Thermonema lapsum ATCC 43542    -   Psychroserpens burtonensis ATCC 700359    -   Gelidibacter algens ATCC 700364    -   Dyadobacter fermentans ATCC 700827    -   Flammeovirga aprica NBRC 15941    -   Spirosoma linguale DSMZ 74    -   Flectobacillus major DSMZ 103    -   Tenacibaculum maritimum ATCC 43398    -   Rhodotermus marinus DSMZ 4252    -   Zobellia galactanivorans DSMZ 12802    -   Muricauda ruestringensis DSMZ 13258    -   Salegentibacter salegens DSMZ 5424    -   Taxeobacter gelupurpurascens DSMZ 11116    -   Cytophaga hutchinsonii NBRC 15051    -   Marinilabilia salmonicolor NBRC 15948    -   Lewinella cohaerens ATCC 23123    -   Saprospira grandis ATCC 23119    -   Haliscomenobacter hydrossis ATCC 27775

Among the aforementioned strains of microbes, those microbes describedwith FERM numbers have been deposited at the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary (Chuo Dai-6, 1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), and can be furnishedby referring to each number.

Among the aforementioned strains of microbes, those microbes describedwith ATCC numbers have been deposited at the American Type CultureCollection (P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica), and can be furnished by referring to each number.

Among the aforementioned strains of microbes, those microbes describedwith IFO numbers have been deposited at the Institute of Fermentation,Osaka (2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan), and can befurnished by referring to each number.

Among the aforementioned strains of microbes, those microbes describedwith NBRC numbers have been deposited at the NITE Biological ResourceCenter of the National Institute of Technology and Evaluation (5-8Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan), and can befurnished by referring to each number.

Among the aforementioned strains of microbes, those microbes describedwith DSMZ numbers have been deposited at the Deutche Sammlung vonMikroorganismen und Zellkulturen GmbH (German Collection of Microbes andCell Cultures) (Mascheroder Weg 1b, 38124 Braunschweig, Germany), andcan be furnished by referring to each number.

Like the aforementioned strains, those microbes described with FERMnumbers are microbes that were deposited at the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary (ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan).Alcaligenes faecalis FERM P-8460 is a microbe that was deposited on Sep.30, 1985 and assigned the deposit number FERM P-8460. Propionibacteriumshermanii FERM P-9737 is a microbe that was originally deposited on Dec.4, 1987 and control of this organism was subsequently transferred tointernational deposition under the provisions of the Budapest Treaty onJul. 1, 2002 and was assigned the deposit number of FERM BP-8100.Xanthomonas maltophilia FERM BP-5568 is a microbe that was originallydeposited on Jun. 14, 1995 and control of this organism was subsequentlytransferred to international deposition under the provisions of theBudapest Treaty on Jun. 14, 1996. Brevibacterium minutiferuna FERMBP-8277 was internationally deposited under the provisions of BudapestTreaty on Jan. 20, 2002. Escherichia coli FERM BP-8276 was deposited atan international depositary institution under the provisions of BudapestTreaty on Jan. 20, 2002.

Empedobacter brevis strain ATCC 14234 (strain FERM P-18545, strain FERMBP-8113) was deposited at the International Patent Organism Depositaryof the independent administrative corporation, National Institute ofAdvanced Industrial Science and Technology (Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. 1, 2001 and assignedthe deposit number of FERM P-18545. Control of this organism wassubsequently transferred to deposition under the provisions of theBudapest Treaty at the International Patent Organism Depositary of theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology on Jul. 8, 2002 and was assigned thedeposit number of FERM BP-8113 (indication of microbe: Empedobacterbrevis strain AJ 13933).

Sphingobacterium sp. strain AJ 110003 was deposited at the InternationalPatent Organism Depositary of the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology (Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jul. 22, 2002, and wasassigned the deposit number of FERM BP-8124.

Note that the strain AJ 110003 (FERM BP-8124) was identified to be theaforementioned Sphingobacterium sp. by the identification experimentdescribed below. The strain FERM BP-8124 is a Gram-negative rod (0.7 to0.8×1.5 to 2.0 μm) that forms no spore and is not motile. Its coloniesare round with a completely smooth border, contain low protrusions andhave a glossy, light yellow color. The organism grows at 30° C. and iscatalase positive, oxidase positive and negative for the OF test(glucose), and was identified as a bacterium belonging to the genusSphingobacterium based on these properties. Moreover, because of theproperties that it is negative for nitrate reduction, negative forindole production, negative for acid production from glucose, argininedihydrolase negative, urease positive, esculin hydrolysis positive,gelatin hydrolysis negative, β-galactosidase positive, glucoseassimilation positive, L-arabinose assimilation negative, D-mannoseassimilation positive, D-mannitol assimilation negative,N-acetyl-D-glucosamine assimilation positive, maltose assimilationpositive, potassium gluconate assimilation negative, n-capric acidassimilation negative, adipic acid assimilation negative, dl-malic acidassimilation negative, sodium citrate assimilation negative, phenylacetate assimilation negative and cytochrome oxidase positive, it wasdetermined to have properties that are similar to those ofSphingobacterium multivorum or Sphingobacterium spiritivorum. Moreover,although results of analyzing analyses on the homology of the basesequence of the 16S rRNA gene indicate the highest degree of homologywas exhibited with Sphingobacterium multivorum (98.8%), there were wasno strain with which the bacterial strain matched completely.Accordingly, this bacterial strain was therefore identified asSphingobacterium sp.

As these microbes, either wild strains or mutant strains can be used orrecombinant strains induced by cell fusion or genetic techniques such asgenetic manipulation can be used.

To obtain microbial cells of such microbes, the microbes can be culturedand grown in a suitable medium. There is no particular restriction onthe medium used for this purpose so far as it allows the microbes togrow. This medium may be an ordinary medium containing ordinary carbonsources, nitrogen sources, phosphorus sources, sulfur sources, inorganicions, and organic nutrient sources as necessary.

For example, any carbon source may be used so far as the microbes canutilize it. Specific examples of the carbon source that can be usedinclude sugars such as glucose, fructose, maltose and amylose, alcoholssuch as sorbitol, ethanol and glycerol, organic acids such as fumaricacid, citric acid, acetic acid and propionic acid and their salts,hydrocarbons such as paraffin as well as mixtures thereof.

Examples of nitrogen sources that can be used include ammonium salts ofinorganic acids such as ammonium sulfate and ammonium chloride, ammoniumsalts of organic acids such as ammonium fumarate and ammonium citrate,nitrates such as sodium nitrate and potassium nitrate, organic nitrogencompounds such as peptones, yeast extract, meat extract and corn steepliquor as well as mixtures thereof.

In addition, nutrient sources used in ordinary media, such as inorganicsalts, trace metal salts and vitamins, can also be suitably mixed andused.

There is no particular restriction on culturing conditions, andculturing can be carried out, for example, for about 12 to about 48hours while properly controlling the pH and temperature within a pHrange of 5 to 8 and a temperature range of 15 to 40° C., respectively,under aerobic conditions.

3. Enzymes Used in the Present Invention

In the method for producing peptide according to the present inventiondescribed above, an enzyme which has an ability to selectively linkL-phenylalanine to the α-ester site of an L-aspartic acid-α,β-diesterthrough a peptide bond is used. In the method for producing peptideaccording to the present invention, the enzyme is not limited by itsorigination and procuring method so far as it has such an activity.Hereinafter, purification of enzymes used in the present invention andutilization of techniques of genetic engineering will be explained.

(3-1) Microbes Having an Enzyme Which can be Used for the ProductionMethod of the Present Invention

As microbes which produce an enzyme of the present invention, all themicrobes that have an ability to produce anα-L-aspartyl-L-phenylalanine-β-ester from an L-aspartic acid-α,β-diesterand L-phenylalanine can be used. The microbes include bacteria and thelike that belong to genera selected from the group consisting ofAeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium,Escherichia, Empedobacter, Flavobacterium, Microbacterium,Propionibacterium, Brevibacillus, Paenibacillus, Pseudomonas, Serratia,Stenotrophomonas, Sphingobacterium, Streptomyces, Xanthomonas,Williopsis, Candida, Geotrichum, Pichia, Saccharomyces, Torulaspora,Cellulophaga, Weeksella, Pedobacter, Persicobacter, Flexithrix,Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens,Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobacillus,Tenacibaculum, Rhodotermus, Zobellia, Muricauda, Salegentibacter,Taxeobacter, Cytophaga, Marinilabilia, Lewinella, Saprospira, andHaliscomenobacter. More specifically, the microbes include Empedobacterbrevis ATCC 14234 (FERM P-18545 strain, FERM BP-8113 strain (Depositaryinstitution: the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, Address of depositary institution: ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan,International deposit transfer date: Jul. 8, 2002)), Sphingobacteriumsp. FERM BP-8124 strain(Depositary institution: the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary,Address of depositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, Japan, International deposit date: Jul. 22,2002), Pedobacter heparinus IFO 12017(Depositary institution: theInstitute of Fermentation, Osaka; 2-17-85 Jusanbon-cho, Yodogawa-ku,Osaka-shi, Japan), Taxeobacter gelupurpurascens DSMZ 11116(Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig,Germany), Cyclobacterium marinum ATCC 25205(Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America), andPsychroserpens burtonensis ATCC 700359 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America) and soforth. Empedobacter brevis ATCC 14234 strain (FERM P-18545 strain, FERMBP-8113 strain (Depositary institution: the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, Address ofdepositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan, International deposit transfer date: Jul. 8, 2002))and Sphingobacterium sp. FERM BP-8124 strain(Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit date:Jul. 22, 2002), Pedobacter heparinus IFO 12017 strain (Depositaryinstitution: the Institute of Fermentation, Osaka; 2-17-85 Jusanbon-cho,Yodogawa-ku, Osaka-shi, Japan), Taxeobacter gelupurpurascens DSMZ 11116strain (Depositary institution; the Deutche Sammlung von Mikroorganismenund Zellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany), Cyclobacterium marinum ATCC 25205 strain(Depositary institution; the American Type Culture Collection, addressof depositary institution; P.O. Box 1549, Manassas, Va. 20110, theUnited States of America), and Psycloserpens burtonensis ATCC 700359strain (Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America) and the like are microbes selected by thepresent inventors as a result of search of enzyme producing microbeswhich produce an α-L-aspartyl-L-phenylalanine-β-ester from an L-asparticacid-α,β-diester and L-phenylalanine at high yield.

(3-2) Purification of Enzyme

As was previously mentioned, the peptide-forming enzyme used in thepresent invention can be purified from bacteria belonging to, forexample, the genus Empedobacter. A method for isolating and purifying apeptide-forming enzyme from Empedobacter brevis is explained as anexample of purification of the enzyme.

First, a microbial cell extract is prepared from microbial cells ofEmpedobacter brevis, for example, the strain FERM BP-8113 (Depositaryinstitution: the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, Address of depositary institution: ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan,International deposit transfer date: Jul. 8, 2002) by disrupting thecells using a physical method such as ultrasonic crushing or anenzymatic method using a cell wall-dissolving enzyme and removing theinsoluble fraction by centrifugal separation and so forth. Thepeptide-producing enzyme can then be purified by fractionating the cellextract obtained in the above manner by combining ordinary proteinpurification methods such as anion exchange chromatography, cationexchange chromatography or gel filtration chromatography.

An example of a carrier for use in anion exchange chromatography isQ-Sepharose HP (manufactured by Amersham). The enzyme is recovered inthe non-adsorbed fraction under conditions of pH 8.5 when the cellextract containing the enzyme is allowed to pass through a column packedwith the carrier.

An example of a carrier for use in cation exchange chromatography isMonoS HR (manufactured by Amersham). After adsorbing the enzyme onto thecolumn by allowing the cell extract containing the enzyme to passthrough a column packed with the carrier and then washing the column,the enzyme is eluted with a buffer solution having a high saltconcentration. At that time, the salt concentration may be sequentiallyincreased or a concentration gradient may be applied. For example, inthe case of using MonoS HR, the enzyme adsorbed onto the column iseluted at an NaCl concentration of about 0.2 to about 0.5 M.

The enzyme purified in the manner described above can then be furtheruniformly purified by gel filtration chromatography and so forth. Anexample of the carrier for use in gel filtration chromatography isSephadex 200 pg (manufactured by Amersham).

In the aforementioned purification procedure, the fraction containingthe enzyme can be verified by assaying the peptide-forming activity ofeach fraction according to the method indicated in the examples to bedescribed later. The internal amino acid sequence of the enzyme purifiedin the manner described above is shown in SEQ ID NO: 1 and SEQ ID NO: 2of the Sequence Listing.

(3-3) Isolation DNA, Production of Transformant and Purification ofPeptide-forming Enzyme

(3-3-1) Isolation of DNA

The inventors of the present invention first succeeded in isolating onetype of DNA of a peptide-forming enzyme that can be used in the peptideproduction method of the present invention from Empedobacter brevisstrain FERM BP-8113 (Depositary institution: the independentadministrative corporation, National institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary,Address of depositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, Japan, International deposit transfer date:Jul. 8, 2002).

A DNA having a base sequence consisting of bases numbers 61 to 1908 ofthe base sequence described in SEQ ID NO: 5, which is a DNA of thepresent invention, was isolated from Empedobacter brevis strain FERMBP-8113 (Depositary institution: the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, Address ofdepositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan, International deposit transfer date: Jul. 8, 2002).The DNA having the base sequence consisting of bases numbers 61 to 1908is a code sequence (CDS) portion. In the base sequence consisting ofbases numbers 61 to 1908 is contained a signal sequence region and amature protein region. The signal sequence region is a region thatconsists of bases numbers 61 to 126, while the mature protein region isa region that consists of bases numbers 127 to 1908. Namely, the presentinvention provides both a gene for a peptide-forming enzyme protein thatcontains a signal sequence, and a gene for a peptide-forming enzymeprotein in the form of a mature protein. The signal sequence containedin the sequence described in SEQ ID NO: 5 is a kind of leader sequence.The main function of a leader peptide encoded by the leader sequence ispresumed to be excretion from inside the cell membrane to outside thecell membrane. The protein encoded by bases numbers 127 to 1908, namelythe-ester site excluding the leader peptide, is presumed to be a matureprotein and exhibit a high degree of peptide-forming activity.

The DNA consisting of the base sequence that consists of bases numbers61 to 1917 described in SEQ ID NO: 11, which is also a DNA of thepresent invention, was isolated from Sphingobacterium sp. strain FERMBP-8124 (Depositary institution: the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, Address ofdepositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan, International deposit date: Jul. 22, 2002). The DNAconsisting of the base sequence that consists of bases numbers 61 to1917 is a code sequence (CDS) portion. In the base sequence consistingof bases numbers 61 to 1917, a signal sequence region and a matureprotein region are contained. The signal sequence region is a regionthat consists of bases numbers 61 to 120, while the mature proteinregion is a region that consists of bases numbers 121 to 1917. Namely,the present invention provides both a gene for a peptide-forming enzymeprotein that contains a signal sequence, and a gene for apeptide-forming enzyme protein in the form of a mature protein. Thesignal sequence contained in the sequence described in SEQ ID NO: 11 isa kind of leader sequence. The main function of a leader peptide encodedby the leader sequence is presumed to be excretion from inside the cellmembrane to outside the cell membrane. The protein encoded by basesnumbers 121 to 1917, namely the portion excluding the leader peptide, ispresumed to be a mature protein and exhibit a high degree ofpeptide-forming activity.

The DNA consisting of the base sequence that consists of bases numbers61 to 1935 described in SEQ ID NO: 17, which is also a DNA of thepresent invention, was isolated from Pedobacter heparinus strain IFO12017 (Depositary institution: the Institute of Fermentation, Osaka;2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan). The DNA consistingof the base sequence that consists of bases numbers 61 to 1935 describedin SEQ ID NO:17 is a code sequence (CDS) portion. In the base sequenceconsisting of bases numbers 61 to 1935, a signal sequence region and amature protein region are contained. The signal sequence region is aregion that consists of bases numbers 61 to 126, while the matureprotein region is a region that consists of bases numbers 127 to 1935.Namely, the present invention provides both a gene for a peptide-formingenzyme protein that contains a signal sequence, and a gene for apeptide-forming enzyme protein in the form of a mature protein. Thesignal sequence contained in the sequence described in SEQ ID NO: 17 isa kind of leader sequence. The main function of a leader peptide encodedby the leader sequence is presumed to be excretion from inside the cellmembrane to outside the cell membrane. The protein encoded by basesnumbers 127 to 1935, namely the portion excluding the leader peptide, ispresumed to be a mature protein and exhibit a high degree ofpeptide-forming activity.

The DNA consisting of the base sequence that consists of bases numbers61 to 1995 described in SEQ ID NO: 22, which is also a DNA of thepresent invention, was isolated from Taxeobacter gelupurpurascens DSMZ11116 (Depositary institution; the Deutche Sammlung von Mikroorganismenund Zellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany). The DNA consisting of the base sequence thatconsists of bases numbers 61 to 1995 described in SEQ ID NO:22 is a codesequence (CDS) portion. In the base sequence consisting of bases numbers61 to 1995, a signal sequence region and a mature protein region arecontained. The signal sequence region is a region that consists of basesnumbers 61 to 126, while the mature protein region is a region thatconsists of bases numbers 127 to 1995. Namely, the present inventionprovides both a gene for a peptide-forming enzyme protein that containsa signal sequence, and a gene for a peptide-forming enzyme protein inthe form of a mature protein. The signal sequence contained in thesequence described in SEQ ID NO: 22 is a kind of leader sequence. Themain function of a leader peptide encoded by the leader sequence ispresumed to be excretion from inside the cell membrane to outside thecell membrane. The protein encoded by bases numbers 127 to 1995, namelythe portion excluding the leader peptide, is presumed to be a matureprotein and exhibit a high degree of peptide-forming activity.

The DNA consisting of the base sequence that consists of bases numbers29 to 1888 described in SEQ ID NO: 24, which is also a DNA of thepresent invention, was isolated from Cyclobacterium marinum ATCC 25205(Depositary institution; the American Type Culture Collection, addressof depositary institution; P.O. Box 1549, Manassas, Va. 20110, theUnited States of America). The DNA consisting of the base sequence thatconsists of bases numbers 29 to 1888 described in SEQ ID NO:24 is a codesequence (CDS) portion. In the base sequence consisting of bases numbers29 to 1888, a signal sequence region and a mature protein region arecontained. The signal sequence region is a region that consists of basesnumbers 29 to 103, while the mature protein region is a region thatconsists of bases numbers 104 to 1888. Namely, the present inventionprovides both a gene for a peptide-forming enzyme protein that containsa signal sequence, and a gene for a peptide-forming enzyme protein inthe form of a mature protein. The signal sequence contained in thesequence described in SEQ ID NO: 24 is a kind of leader sequence. Themain function of a leader peptide encoded by the leader sequence ispresumed to be excretion from inside the cell membrane to outside thecell membrane. The protein encoded by bases numbers 104 to 1888, namelythe portion excluding the leader peptide, is presumed to be a matureprotein and exhibit a high degree of peptide-forming activity.

The DNA consisting of the base sequence that consists of bases numbers61 to 1992 described in SEQ ID NO: 26, which is also a DNA of thepresent invention, was isolated from Psychroserpens burtonensis ATCC700359 (Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America). The DNA consisting of the base sequencethat consists of bases numbers 61 to 1992 described in SEQ ID NO:26 is acode sequence (CDS) portion. In the base sequence consisting of basesnumbers 61 to 1992, a signal sequence region and a mature protein regionare contained. The signal sequence region is a region that consists ofbases numbers 61 to 111, while the mature protein region is a regionthat consists of bases numbers 112 to 1992. Namely, the presentinvention provides both a gene for a peptide-forming enzyme protein thatcontains a signal sequence, and a gene for a peptide-forming enzymeprotein in the form of a mature protein. The signal sequence containedin the sequence described in SEQ ID NO: 26 is a kind of leader sequence.The main function of a leader peptide encoded by the leader sequence ispresumed to be excretion from inside the cell membrane to outside thecell membrane. The protein encoded by bases numbers 112 to 1992, namelythe portion excluding the leader peptide, is presumed to be a matureprotein and exhibit a high degree of peptide-forming activity.

Furthermore, the various gene recombination techniques indicated belowcan be carried out in accordance with the descriptions in MolecularCloning, 2nd edition, Cold Spring Harbor Press (1989) and otherpublications.

A DNA encoding an enzyme that can be used in the present invention canbe acquired by polymerase chain reaction (PCR, refer to White, T. J. etal., Trends Genet., 5, 185 (1989)) or hybridization from a chromosomalDNA or a DNA library of Empedobacter brevis, Sphingobacterium sp.,Pedobacter heparinus, Taxeobacter gelupurpurascens, Cyclobacteriummarinum, or Psychroserpens burtonensis. Primers used in PCR can bedesigned based on the internal amino acid base sequences determined onthe basis of purified peptide-forming enzyme as explained in theprevious section (3). In addition, since the base sequences of thepeptide-forming enzyme genes (SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO:17,SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26) have been identified bythe present invention, primers or hybridization probes can be designedon the basis of these base sequences, and the gene can be isolated usingthe probes. If primers having sequences corresponding to the5′-nontranslated region and 3′-nontranslated region, respectively, areused as PCR primers, the full-length encoded region of the enzyme can beamplified. Taking as an example the case of amplifying a regioncontaining both the leader sequence and a mature protein encoding regionas described in SEQ ID NO: 5, specific examples of primers include aprimer having a base sequence of the region upstream of base number 61in SEQ ID NO: 5 for the 5′-side primer, and a primer having a sequencecomplementary to the base sequence of the region downstream of basenumber 1908 for the 3′-side primer.

Primers can be synthesized, for example, according to ordinary methodsusing the phosphoamidite method (refer to Tetrahedron Letters (1981),22, 1859) by use of the Model 380B DNA Synthesizer manufactured byApplied Biosystems. The PCR reaction can be carried out, for example, byusing the Gene Amp PCR System 9600 (Perkin-Elmer) and the Takara LA PCRIn Vitro Cloning Kit (Takara Shuzo) in accordance with the methodspecified by the supplier such as the manufacturer.

A DNA that encodes an enzyme that can be used in the peptide productionmethod of the present invention, regardless of whether the DNA containsa leader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 5 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizesa DNA consisting of a base sequence complementary to the CDS describedin SEQ ID NO: 5 of the Sequence Listing or with a probe prepared fromthe base sequence under stringent conditions and encodes a proteinhaving peptide-forming activity from a DNA encoding the mutant enzyme orcells that possess that DNA.

The DNA of the present invention, regardless of whether it contains aleader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 11 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizesa DNA consisting of a base sequence complementary to the CDS describedin SEQ ID NO: 11 of the Sequence Listing or with a probe prepared fromthe base sequence under stringent conditions and encodes a proteinhaving peptide-forming activity from a DNA encoding the mutant enzyme orcells that possess that DNA.

The DNA of the present invention, regardless of whether it contains aleader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 17 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizeswith a DNA consisting of a base sequence complementary to the CDSdescribed in SEQ ID NO: 17 of the Sequence Listing or with a probeprepared from the base sequence under stringent conditions and encodes aprotein having peptide-forming activity from a DNA encoding the mutantenzyme or cells that possess that DNA.

The DNA of the present invention, regardless of whether it contains aleader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 22 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizeswith a DNA consisting of a base sequence complementary to the CDSdescribed in SEQ ID NO: 22 of the Sequence Listing or with a probeprepared from the base sequence under stringent conditions and encodes aprotein having peptide-forming activity from a DNA encoding the mutantenzyme or cells that possess that DNA.

The DNA of the present invention, regardless of whether it contains aleader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 24 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizeswith a DNA consisting of a base sequence complementary to the CDSdescribed in SEQ ID NO: 24 of the Sequence Listing or with a probeprepared from the base sequence under stringent conditions and encodes aprotein having peptide-forming activity from a DNA encoding the mutantenzyme or cells that possess that DNA.

The DNA of the present invention, regardless of whether it contains aleader sequence or not, includes a DNA that is substantially identicalto the DNA consisting of the CDS described in SEQ ID NO: 26 of theSequence Listing. Namely, a DNA substantially identical to the DNA ofthe present invention can be obtained by isolating a DNA that hybridizeswith a DNA consisting of a base sequence complementary to the CDSdescribed in SEQ ID NO: 26 of the Sequence Listing or with a probeprepared from the base sequence under stringent conditions and encodes aprotein having peptide-forming activity from a DNA encoding the mutantenzyme or cells that possess that DNA.

A probe can be produced, for example, in accordance with establishedmethods based on, for example, the base sequence described in SEQ ID NO:5 of the Sequence Listing. In addition, a method for isolating a targetDNA by using a probe to find a DNA that hybridizes with the probe mayalso be carried out in accordance with established methods. For example,a DNA probe can be produced by amplifying a base sequence cloned in aplasmid or phage vector, cleaving the base sequence desired to be usedas a probe with a restriction enzyme and then extracting the desiredbase sequence. The portion to be cleaved out can be adjusted dependingon the target DNA.

The term “under a stringent condition” as used herein refers to acondition under which a so-called specific hybrid is formed but nonon-specific hybrid is formed. It is difficult to precisely express thiscondition in numerical values. For example, mention may be made of acondition under which DNAs having high homologies, for example, 50% ormore, preferably 80% or more, more preferably 90% or more, hybridizewith each other and DNAs having lower homologies than these do nothybridize with each other, or ordinary conditions for rinse in Southernhybridization under which hybridization is performed at 60° C. in a saltconcentration corresponding 1×SSC and 0.1% SDS, preferably 0.1×SSC and0.1% SDS. Although the genes that hybridize under such conditionsinclude those genes in which stop codons have occurred at certainlocations along their sequences or which have lost activity due to amutation in the active center, these can be easily removed by ligatingthem to a commercially available expression vector, expressing them in asuitable host, and assaying the enzyme activity of the expressionproduct using a method to be described later.

However, in the case of a base sequence that hybridizes under stringentconditions as described above, it is preferable that the protein encodedby that base sequence retains about a half or more, preferably 80% ormore, and more preferably 90% or more, of the enzyme activity of theprotein having the amino acid sequence encoded by the original basesequence serving as the base be retained under conditions of 50° C. andpH 8. For example, when explained for on the case of, for example, abase sequence that hybridizes under stringent conditions with a DNA thathas a base sequence complementary to the base sequence consisting ofbases numbers 127 to 1908 of the base sequence described in SEQ ID NO:5, it is preferable that the protein encoded by that base sequenceretains about a half or more, preferably 80% or more, and morepreferably 90% or more, of the enzyme activity of the protein having anamino acid sequence that consists of amino acid residues numbers 23 to616 of the amino acid sequence described in SEQ ID NO: 6 underconditions of 50° C. and pH 8.

An amino acid sequence encoded by the CDS described in SEQ ID NO: 5 ofthe Sequence Listing is shown in SEQ ID NO: 6 of the Sequence Listing.An amino acid sequence encoded by the CDS described in SEQ ID NO: 11 ofthe Sequence Listing is shown in SEQ ID NO: 12 of the Sequence Listing.An amino acid sequence encoded by the CDS described in SEQ ID NO.: 17 ofthe Sequence Listing is shown in SEQ ID NO: 18 of the Sequence Listing.An amino acid sequence encoded by the CDS described in SEQ ID NO: 22 ofthe Sequence Listing is shown in SEQ ID NO: 23 of the Sequence Listing.An amino acid sequence encoded by the CDS described in SEQ ID NO: 24 ofthe Sequence Listing is shown in SEQ ID NO: 25 of the Sequence Listing.An amino acid sequence encoded by the CDS described in SEQ ID NO: 26 ofthe Sequence Listing is shown in SEQ ID NO: 27 of the Sequence Listing.

The entire amino acid sequence described in SEQ ID NO: 6 contains aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 22 constituting the leader peptide, and amino acid residuesnumbers 23 to 616 constituting the mature protein region.

The entire amino acid sequence described in SEQ ID NO: 11 includes aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 20 constituting the leader peptide, and amino acid residuesnumbers 21 to 619 constituting the mature protein region.

The entire amino acid sequence described in SEQ ID NO: 18 contains aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 22 constituting the leader peptide, and amino acid residuesnumbers 23 to 625 constituting the mature protein region.

The entire amino acid sequence described in SEQ ID NO: 23 contains aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 22 constituting the leader peptide, and amino acid residuesnumbers 23 to 645 constituting the mature protein region.

The entire amino acid sequence described in SEQ ID NO: 25 contains aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 25 constituting the leader peptide, and amino acid residuesnumbers 26 to 620 constituting the mature protein region.

The entire amino acid sequence described in SEQ ID NO: 27 contains aleader peptide and a mature protein region, with amino acid residuesnumbers 1 to 17 constituting the leader peptide, and amino acid residuesnumbers 18 to 644 constituting the mature protein region.

The protein encoded by the DNA of the present invention is a protein inwhich the mature protein has peptide-forming activity, and a DNA thatencodes a protein substantially identical to a protein having the aminoacid sequence described in SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18,SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27 of the Sequence Listing,regardless of whether it contains a leader peptide or not, is alsoincluded in the DNA of the present invention. (Note that, base sequencesare specified from amino acid sequences in accordance with the codes ofthe universal codons.) Namely, the present invention provides DNAs thatencode proteins indicated in (A) to (X) below:

-   -   (A) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 616 of an amino acid sequence        described in SEQ ID NO: 6 of the Sequence Listing,    -   (B) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 616 of the amino        acid sequence described in SEQ ID NO: 6 of the Sequence Listing,        and having activity to selectively linking L-phenylalanine to an        α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond,    -   (C) a protein having the amino acid sequence consisting of amino        acid residue numbers 21 to 619 of an amino acid sequence        described in SEQ ID NO: 12 of the Sequence Listing,    -   (D) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residue numbers 21 to 619 of the amino        acid sequence described in SEQ ID NO: 12 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (E) a protein having the amino acid sequence consisting of amino        acid residues numbers 23 to 625 of an amino acid sequence        described in SEQ ID NO: 18 of the Sequence Listing,    -   (F) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 625 of the amino        acid sequence described in SEQ ID NO: 18 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (G) a protein having an amino acid sequence consisting of amino        acid residues numbers 23 to 645 of an amino acid sequence        described in SEQ ID NO: 23 of the Sequence Listing,    -   (H) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 23 to 645 of the amino        acid sequence described in SEQ ID NO: 23 of the Sequence        Listing, and activity to selectively linking L-phenylalanine to        an α-ester site of the L-aspartic acid-α,β-diester through a        peptide bond,    -   (I) a protein having an amino acid sequence consisting of amino        acid residues numbers 26 to 620 of an amino acid sequence        described in SEQ ID NO: 25 of the Sequence Listing,    -   (J) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 26 to 620 of the amino        acid sequence described in SEQ ID NO: 25 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (K) a protein having an amino acid sequence consisting of amino        acid residues numbers 18 to 644 of an amino acid sequence        described in SEQ ID NO: 27 of the Sequence Listing,    -   (L) a protein having an amino acid sequence including        substitution, deletion, insertion, addition, and/or inversion of        one or a plurality of amino acids in the amino acid sequence        consisting of amino acid residues numbers 18 to 644 of the amino        acid sequence described in SEQ ID NO: 27 of the Sequence        Listing, and having activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (M) a protein having an amino acid sequence described in SEQ ID        NO: 6 of the Sequence Listing,    -   (N) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 6 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (O) a protein having the amino acid sequence described in SEQ ID        NO: 12 of the Sequence Listing,    -   (P) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 12 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (Q) a protein having an amino acid sequence described in SEQ ID        NO: 18 of the Sequence Listing,    -   (R) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 18 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (S) a protein having an amino acid sequence described in SEQ ID        NO: 23 of the Sequence Listing,    -   (T) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 23 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (U) a protein having an amino acid sequence described in SEQ ID        NO: 25 of the Sequence Listing,    -   (V) a protein containing a mature protein region, having an        amino acid sequence including substitution, deletion, insertion,        addition, and/or inversion of one or a plurality of amino acids        in the amino acid sequence described in SEQ ID NO: 25 of the        Sequence Listing, and activity to selectively linking        L-phenylalanine to an α-ester site of the L-aspartic        acid-α,β-diester through a peptide bond,    -   (W) a protein having an amino acid sequence described in SEQ ID        NO: 27 of the Sequence Listing, and    -   (X) a protein containing a mature protein region, having an        amino acid sequence in the amino acid sequence described in SEQ        ID NO: 27 of the Sequence Listing, and having activity to        selectively linking L-phenylalanine to an α-ester site of the        L-aspartic acid-α,β-diester through a peptide bond.

Here, although the meaning of the term “a plurality of” varies dependingon the locations and types of the amino acid residues in thethree-dimensional structure of the protein, it is within a range thatdoes not significantly impair the three-dimensional structure andactivity of the protein of the amino acid residues, and is specifically2 to 50, preferably 2 to 30, and more preferably 2 to 10. However, inthe case of amino acid sequences including substitution, deletion,insertion, addition, and/or inversion of one or a plurality of aminoacids in amino acid sequences of the proteins of (B), (D), (F), (H),(J), (L), (N), (P), (R), (T), (V) or (X), it is preferable that theproteins retain about half or more, more preferably 80% or more, andeven more preferably 90% or more of the enzyme activity of the proteinsin the state where no mutation is included, under conditions of 50° C.and pH 8. For example, explanation will be made in the case of (B); inthe case of the amino acid sequence (B) including substitution,deletion, insertion, addition, and/or inversion of one or a plurality ofamino acids in an amino acid sequence described in SEQ ID NO: 6 of theSequence Listing, it is preferable that this protein retains about halfor more, more preferably 80% or more, and even more preferably 90% ormore of the enzyme activity of the protein having the amino acidsequence described in SEQ ID NO: 6 of the Sequence Listing, underconditions of 50° C. and pH 8.

A mutation of an amino acid like that indicated in the aforementioned(B) and so forth is obtained by modifying the base sequence so that anamino acid of a specific-ester site in the present enzyme gene issubstituted, deleted, inserted or added by, for example,-estersite-directed mutagenesis. In addition, a modified DNA like thatdescribed above can also be acquired by mutagenesis treatment known inthe art. Mutagenesis treatment refers to, for example, a method in whicha DNA encoding the present enzyme is treated in vitro with hydroxylamineand so forth, as well as a method in which Escherichia bacteria thatpossess a DNA encoding the present enzyme are treated by a mutagennormally used in artificial mutagenesis, such as ultravioletirradiation, N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.

In addition, naturally-occurring mutations such as differencesattributable to a microbe species or strain are also included in thebase substitution, deletion, insertion, addition and/or inversiondescribed above. By expressing a DNA having such a mutation in suitablecells and investigating the enzyme activity of the expression product, aDNA can be obtained that encodes a protein substantially identical tothe protein described in SEQ ID NO: 6, 12, 18, 23, 25 or 27 of theSequence Listing.

(3-3-2) Preparation of Transformants and Production of Peptide-FormingEnzymes

Peptide-forming enzymes that can be used in the method of producing apeptide according to the present invention can be produced byintroducing the DNA explained in (3-3-1) above into a suitable host andexpressing the DNA in that host.

With respect to hosts for expressing a protein specified by the DNA,examples of the hosts that can be used include various prokaryotic cellsincluding Escherichia bacteria such as Escherichia coli, Empedobacterbacteria, Sphingobacterium bacteria, Flavobacterium bacteria andBacillus subtilis, as well as various eukaryotic cells includingSaccharomyces cerevisiae, Pichia stipitis and Aspergillus oryzae.

A recombinant DNA used to introduce a DNA into a host can be prepared byinserting the DNA to be introduced into a vector corresponding to thetype of host in which the DNA is to be expressed, in such a form thatthe protein encoded by that DNA can be expressed. In the case where apromoter unique to a peptide-forming enzyme gene of Empedobacter brevisand so forth functions in the host cells, the promoter can be used as apromoter for expressing the DNA of the present invention. In addition,another promoter that acts in the host cells may be ligated to the DNAof the present invention, and the DNA may be expressed under the controlof the promoter as necessary.

Examples of transformation methods for introducing a recombinant DNAinto host cells include the method of D. M. Morrison (see Methods inEnzymology, 68, 326 (1979)) or the method in which DNA permeability isincreased by treating receptor microbial cells with calcium chloride(see Mandel, H. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

In the case of mass production of a protein using recombinant DNAtechnology, conjugating the protein within a transformant that producesthe protein to form an inclusion body of protein is also a preferablemode for carrying out the present invention. Advantages of thisexpression and production method include protection of the targetprotein from digestion by proteases present within the microbial cells,and simple and easy purification of the target protein by disrupting themicrobial cells followed by centrifugal separation and so forth.

The inclusion body of protein obtained in this manner is solubilizedwith a protein denaturant and the protein is converted to a properlyfolded, physiologically active protein through an activity regenerationprocedure that consists primarily of removal of the denaturant. Thereare numerous examples of this, including regeneration of the activity ofhuman interleukin-2 (see Japanese Patent Application Laid-openPublication No. S61-257931).

To obtain an active protein from inclusion bodies of, a series ofprocedures including solubilization and activity regeneration arerequired, and the procedure is more complex than in the case ofproducing the active protein directly. However, in the case of producinga protein that has a detrimental effect on microbial growth in largevolumes within microbial cells, that effect can be suppressed byaccumulating the proteins in the form of inclusion bodies of inactiveprotein within the microbial cells.

Examples of mass production methods for producing a target protein inthe form of inclusion bodies include a method in which a target proteinis expressed independently under the control of a powerful promoter, anda method in which a target protein is expressed in the form of a fusedprotein with a protein that is known to be expressed in a large volume.

Hereinafter, the present invention will be explained more specificallytaking as an example a method for producing transformed Escherichia coliand using that transformed microbe to produce a peptide-forming enzyme.Furthermore, in the case of producing peptide-forming enzyme in amicrobe such as Escherichia coli, a DNA that encodes a precursor proteincontaining a leader sequence may be incorporated or a DNA that consistsonly of a mature protein region that does not contain a leader sequencemay be incorporated, and the DNA can be suitably selected for theprotein encoding sequence depending on the production conditions, form,usage conditions and so forth of the enzyme to be produced.

Promoters normally used in the production of heterogeneous proteins inEscherichia. coli can be used as a promoter for expressing a DNAencoding a peptide-forming enzyme. Examples of such promoters include T7promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambdaphage PR promoter, PL promoter and other powerful promoters. Inaddition, examples of vectors that can be used include pUC19, pUC18,pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118,pMW219, and pMW218. Besides, vectors of phage DNA can also be used.Moreover, expression vectors that contain promoters and are capable ofexpressing an inserted DNA sequence can be used.

To produce peptide-forming enzyme in the form of a fused proteininclusion body, a gene that encodes another protein, and preferably ahydrophilic peptide, is ligated upstream or downstream of thepeptide-forming enzyme gene to obtain a fused protein gene. The genethat encodes another protein in this manner may be any gene thatincreases the amount of the fused protein accumulated and enhances thesolubility of the fused protein after the denaturation and regenerationsteps. Examples of candidates for such genes include T7 gene 10,β-galactosidase gene, dehydrofolate reductase gene, γ-interferon gene,interleukin-2 gene and prochymosin gene.

When these genes are ligated to the genes that encode peptide-formingenzymes, the genes are ligated so that reading frames of codons areconsistent. It is recommended that the genes be ligated at a properrestriction enzyme-ester site or a synthetic DNA having a propersequence be utilized.

Further, to increase a production amount of the peptide-forming enzyme,it is preferable in some cases that a terminator, which is atranscription terminating sequence, be ligated to downstream of thefusion protein gene. The terminator includes, for example, a T7terminator, an fd phage terminator, a T4 terminator, a tetracyclineresistant gene terminator, and an Escherichia coli trpA gene terminator.

As the vectors for introducing a gene that encodes a peptide-formingenzyme or a fused protein between the peptide-forming enzyme and anotherprotein in Escherichia coli are preferred so-called multi-copy typevectors, examples of which include a plasmid having a replicator derivedfrom ColE1, for example, a pUC-based plasmid, and a pBR322-based plasmidor derivatives thereof. The “derivatives” as used herein refer to thoseplasmids that are subjected modification by substitution, deletion,insertion, addition and/or inversion of bases. Note that themodification as used herein includes modifications by a mutationtreatment with a mutagen or UV irradiation, or modifications byspontaneous mutation.

To screen transformants, it is preferable that the vectors have markerssuch as an ampicillin resistant gene. As such plasmids are commerciallyavailable expression vectors having potent promoters (a pUC-based vector(manufactured by Takara Shuzo, Co., Ltd.), pRROK-based vector(manufactured by Clonetech Laboratories, Inc.), pKK233-2 (manufacturedby Clonetech Laboratories, Inc.) and so forth.

A recombinant DNA is obtained by ligating a DNA fragment to a vectorDNA. In this case, a promoter, a gene encoding L-amino acid amidehydrolase or a fused protein consisting of an L-amino acid amidehydrolase and another protein, and depending on the case, a terminatorare ligated in that order.

When Escherichia coli is transformed using the recombinant DNA and theresulting Escherichia coli is cultured, a peptide-forming enzyme or afused protein consisting of the peptide-forming enzyme and anotherprotein is expressed and produced. Although a strain that is normallyused in the expression of a heterogeneous gene can be used as a host tobe transformed, Escherichia coli strain JM109, for example, ispreferable. Methods for carrying out transformation and methods forscreening out transformants are described in Molecular Cloning, 2ndEdition, Cold Spring Harbor Press (1989) and other publications.

In the case of expressing a peptide-forming enzyme in the form of afusion protein, the peptide-forming enzyme may be cleaved out using arestriction protease that uses a sequence not present in thepeptide-forming enzyme, such as blood coagulation factor Xa orkallikrein, as the recognition sequence.

A medium normally used for culturing Escherichia coli, such asM9-casamino acid medium or LB medium, may be used as a productionmedium. In addition, culturing conditions and production inductionconditions are suitably selected according to the marker of the vectorused, promoter, type of host microbe and so forth.

The following method can be used to recover the peptide-forming enzymeor fused protein consisting of the peptide-forming enzyme and anotherprotein. If the peptide-forming enzyme or its fused protein has beensolubilized within the microbial cells, after recovering the microbialcells, the microbial cells are crushed or lysed so that they can be usedas a crude enzyme liquid. Moreover, the peptide-forming enzyme or itsfused protein can be purified prior to use by ordinary techniques suchas precipitation, filtration or column chromatography as necessary. Inthis case, a purification method can also be used that uses an antibodyof the peptide-forming enzyme or its fused protein.

In the case where protein inclusion bodies are formed, the inclusionbodies are solubilized with a denaturant. They may be solubilizedtogether with the microbial cell protein. However, in consideration ofthe following purification procedure, the inclusion bodies arepreferably taken out and then solubilized. Conventionally known methodsmay be used to recover the inclusion bodies from the microbial cells.For example, inclusion bodies can be recovered by crushing the microbialcells followed by centrifugal separation. Examples of denaturantscapable of solubilizing inclusion bodies include guanidine hydrochloride(for example, 6 M, pH 5 to 8) and urea (for example, 8 M).

A protein having activity is regenerated by removing these denaturantsby dialysis. A Tris-HCl buffer solution or a phosphate buffer solutionand so forth may be used as the dialysis solution to be used indialysis, and the concentration may be, for example, 20 mM to 0.5 M,while the pH may be, for example, 5 to 8.

The protein concentration during the regeneration step is preferablyheld to about 500 μg/ml or less. The dialysis temperature is preferably5° C. or lower to inhibit the regenerated peptide-forming enzyme fromundergoing self-crosslinking. Moreover, in addition to dialysis,dilution or ultrafiltration may be used to remove the denaturants, andit is expected that the activity can be regenerated regardless ofwhichever denaturant is used.

<2>Method of Producing α-L-aspartyl-L-phenylalanine-α-methyl ester

The method of producing α-APM according to the present inventionincludes a first step of synthesizingα-L-aspartyl-L-phenylalanine-β-methyl ester according to the “<1> Methodof producing α-L-aspartyl-L-phenylalanine-β-ester” and a second step ofconverting α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.

Preferred conditions in the first step and the like are as described inthe “<1> Method of producing α-L-aspartyl-L-phenylalanine-β-ester”. Inaddition, the second step can be carried out according to the knownmethod and reference may be made to the method and preferred conditionsdescribed in, for example, Japanese Patent Publication No. H4-41155,etc. By the production method of α-APM according to the presentinvention, α-APM, which is important as a sweetener and the like, can beinexpensively produced at high yield.

EXAMPLES

Hereinafter, the present invention will be explained by examples.However, the present invention is not limited to these examples. Inaddition to confirmation by ninhydrin coloring of thin-filmchromatograms (qualitative), quantitative determinations were made bythe following high-performance liquid chromatography in order to assayproducts. Column: InertsiL ODS-2 (manufactured by GL Science, Inc.),eluate: aqueous phosphate solution containing 5.0 mM sodium1-octanesulfonate (pH 2.1): methanol=100:15 to 50, flow rate: 1.0mL/min, detection: 210 nanometers (nm).

Example 1 Microbes that Produce α-L-aspartyl-L-phenylalanine-β-methylester

50 milliliters (“mL” or “ml”) of a medium (pH 7.0) containing 20 grams(“g”) of glycerol, 5 g of ammonium sulfate, 1 g of potassium dihydrogenphosphate, 3 g of dipotassium hydrogen phosphate, 0.5 g of magnesiumsulfate, 10 g of yeast extract and 10 g of peptone in 1 liter (L) thatwas transferred to a 500 mL Sakaguchi flask and sterilized at 115° C.for 15 minutes (medium 1) was used to culture bacteria and actinomycetesshown in Table 1-1. A slant agar medium (pH 7.0) containing 5 g/L ofglucose, 10 g/L of yeast extract, 10 g/L of peptone, 5 g/L of NaCl and20 g/L of agar in the medium 1 was prepared and a microbe shown in Table1 was cultured on this slant agar medium at 30° C. for 24 hours. Then,one loopful of the microbe was cultured in the medium 1 at 30° C. for 24hours, followed by shake culturing at 30° C. and 120 strokes/min for 17hours. After completion of the culturing, the microbial cells wereseparated from these culture liquids by centrifugation, and suspended in0.1 M borate buffer (pH 9.0) containing 10 mM EDTA to 100 g/L as wetmicrobial cells.

To culture yeast shown in Table 1-1, 50 mL of a medium (pH 6.0)containing 10 g of glucose, 10 g of glycerol, 5 g of ammonium sulfate, 1g of potassium dihydrogen phosphate, 3 g of dipotassium hydrogenphosphate, 0.5 g of magnesium sulfate, 5 g of yeast extract, 5 g of maltextract and 10 g of peptone in 1 L transferred to a 500-mL Sakaguchiflask and sterilized at 115° C. for 15 minutes (medium 2) was used. Aslant agar medium (pH 6.0) containing 5 g/L of glucose, 5 g/L of yeastextract, 5 g/L of malt extract, 10 g/L of peptone, 5 g/L of NaCl and 20g/L agar in the medium 2 was prepared and a yeast shown in Table 1 wascultured on the slant agar medium at 30° C. for 24 hours. Then, oneloopful of the yeast was shake cultured at 30° C. for 24 hours in themedium 2 at 25° C. and 120 strokes/min for 17 hours. After completion ofthe culturing, the microbial cells were separated from these cultureliquids by centrifugation, and suspended in 0.1 M borate buffer (pH 9.0)containing 10 mM EDTA to 100 g/L as wet microbial cells.

The microbes shown in Table 1-2 were cultured as follows. An agar solidmedium (pH 7.2, sterilized at 120° C. for 15 minutes) containing 1 g oftryptone, 1 g of yeast extract and 15 g of agar in 1 L of Daigoartificial sea water SP was used to culture Cellulophaga lytica NBRC14961(Depositary institution; the NITE Biological Resource Center of theNational Institute of Technology and Evaluation, address of depositaryinstitution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken,Japan) or Flexithrix dorotheae NBRC 15987 (Depositary institution; theNITE Biological Resource Center of the National institute of Technologyand Evaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan). Microbial cells ofCellulophaga lytica NBRC 14961 (Depositary institution; the NITEBiological Resource Center of the National Institute of Technology andEvaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan) or Flexithrix dorotheae NBRC15987 (Depositary institution; the NITE Biological Resource Center ofthe National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan) which was seed cultured on this medium at 30° C. for48 hours were applied on the same medium, followed by main culturing at30° C. for 48 hours.

A sheep blood agar medium (Nissui Plate, Nissui Pharmaceutical) was usedto culture Weeksella virosa NBRC 16016 (Depositary institution; the NITEBiological Resource Center of the National Institute of Technology andEvaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan). Microbial cells of Weeksellavirosa NBRC 16016 (Depositary institution; the NITE Biological ResourceCenter of the National Institute of Technology and Evaluation, addressof depositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan) which was seed cultured on this medium at 30° C. for48 hours were applied on the same medium, followed by main culturing at30° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 10 g of peptone, 2 g of yeast extract, 1 g of MgSO₄.7H₂O and15 g of agar in 1 L of distilled water was used to culture Pedobacterheparinus NBRC 12017(Depositary institution; the NITE BiologicalResource Center of the National Institute of Technology and Evaluation,address of depositary institution; 5-8 Kazusa-Kamaashi 2-Chome,Kisarazu-shi, Chiba-ken, Japan). Microbial cells of Pedobacter heparinusNBRC 12017 (Depositary institution; the NITE Biological Resource Centerof the National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan) which was seed cultured on this medium at 30° C. for48 hours were applied on the same medium, followed by main culturing at30° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 0.5 g of KNO₃, 0.1 g of sodium glycerophosphate, 1 g oftrishydroxymethylaminomethane, 5 g of tryptone, 5 g of yeast extract, 15g of agar and 1 ml of a trace element solution in 1 L of Daigoartificial sea water SP was used to culture Persicobacter diffluens NBRC15940 ((Depositary institution; the NITE Biological Resource Center ofthe National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan) Note that the trace element solution contained 2.85 gof H₃BO₄, 1.8 g of MnCl₂.4H₂O, 1.36 g of FeSO₄.7H₂O, 26.9 mg ofCuCl₂.2H₂O, 20.8 mg of ZnCl₂, 40.4 mg of CoCl₂.6H₂O, 25.2 mg ofNa₂MoO₄.2H₂O, and 1.77 g of sodium tartrate). Microbial cells ofPersicobacter diffluens NBRC 15940 (Depositary institution; the NITEBiological Resource Center of the National Institute of Technology. andEvaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan) which was seed cultured on thismedium at 25° C. for 48 hours were applied on the same medium, followedby main culturing at 25° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 3 g of bactocasitone, 1 g of yeast extract, 1.36 g ofCaCl₂.2H₂O and 15 g of agar in 1 L of distilled water was used toculture Chitinophaga pinensis NBRC 15968 (Depositary institution; theNITE Biological Resource Center of the National Institute of Technologyand Evaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan). Microbial cells ofChitinophaga pinensis NBRC 15968(Depositary institution; the NITEBiological Resource Center of the National Institute of Technology andEvaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan) which was seed cultured on thismedium at 25° C. for 48 hours were applied on the same medium, followedby main culturing at 25° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 5 g of peptone, 1 g of yeast extract, 0.2 g of FeSO₄.7H₂O and15 g of agar in 1 L of Daigo artificial sea water SP was used to cultureCyclobacterium marinum ATCC 25205 (Depositary institution; the AmericanType Culture Collection, address of depositary institution; P.O. Box1549, Manassas, Va. 20110, the United States of America). Microbialcells of Cyclobacterium marinum ATCC 25205 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America) whichwas seed cultured on this medium at 25° C. for 48 hours were applied onthe same medium, followed by main culturing at 25° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 1 g of peptone, 1 g of yeast extract, 1 g of glucose and 15 gof agar in 1 L of distilled water was used to culture Runellaslithyformis ATCC 29530 (Depositary institution; the American TypeCulture Collection, address of depositary institution; P.O. Box 1549,Manassas, Va. 20110, the United States of America). Microbial cells ofRunella slithyformis ATCC 29530 (Depositary institution; the AmericanType Culture Collection, address of depositary institution; P.O. Box1549, Manassas, Va. 20110, the United States of America) which was seedcultured in this medium at 25° C. for 48 hours were applied on the samemedium, followed by main culturing at 25° C. for 48 hours.

An agar solid medium (pH 8.2, sterilized at 120° C. for 15 minutes)containing 0.2 g of nitrilotriacetic acid, 2 ml of a 0.03% FeCl₃solution, 0.12 g of CaSO₄.2H₂O, 0.2 g of MgSO₄.7H₂O, 0.016 g of NaCl,0.21 g of KNO₃, 1.4 g of NaNO₃, 0.22 g of Na₂HPO₄, 2 ml of trace elementsolution and 15 g of agar in 1 L of distilled water was used to cultureThermonema lapsum ATCC 43542 ((Depositary institution; the American TypeCulture Collection, address of depositary institution; P.O. Box 1549,Manassas, Va. 20110, the United States of America) it should be notedthat the trace element solution contained 0.5 ml of H₂SO₄, 2.2 g ofMnSO₄, 0.5 g of ZnSO₄, 0.5 g of H₃BO₃, 0.016 g of CuSO₄, 0.025 g ofNa₂MoO₄ and 0.046 g of CoCl₂). Microbial cells of Thermonema lapsum ATCC43542 (Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America) which was seed cultured on this medium at60° C. for 48 hours were applied on the same medium, followed by mainculturing at 25° C. for 48 hours.

Marine Agar 2216 (manufactured by Difco) was used to cultureGelidibacter algens ATCC 700364 (Depositary institution; the AmericanType Culture Collection, address of depositary institution; P.O. Box1549, Manassas, Va. 20110, the United States of America), Lewinellacohaerens ATCC 23123 (Depositary institution; the American Type CultureCollection, address of depositary institution; P.O. Box 1549, Manassas,Va. 20110, the United States of America), Psychroserpens burtonensisATCC 700359 (Depositary institution; the American Type CultureCollection, address of depositary institution; P.O. Box 1549, Manassas,Va. 20110, the United States of America), or Salegentibacter salegensDSMZ 5424 (Depositary institution; the Deutche Sammlung vonMikroorganismen und Zellkulturen GmbH (German Collection of Microbes andCell Cultures, Address of Depositary institution; Mascheroder Weg 1b,38124 Braunschweig, Germany). In the case of Gelidibacter algens ATCC700364 (Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America) or Psychroserpens burtonensis ATCC 700359(Depositary institution; the American Type Culture Collection, addressof depositary institution; P.O. Box 1549, Manassas, Va. 20110, theUnited States of America), microbial cells of Gelidibacter algens ATCC700364, or Psychroserpens burtonensis ATCC 700359 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) which was seed cultured on this medium at 10° C. for 72 hourswere applied, followed by main culturing at 10° C. for 72 hours. In thecase of Lewinella cohaerens ATCC 23123 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America),microbial cells of Lewinella cohaerens ATCC 23123 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) which was seed cultured in this medium at 30° C. for 48 hourswere applied on the same medium, followed by main culturing at 30° C.for 48 hours. In the case of Salegentibacter salegens DSMZ 5424(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany), microbial cells of Salegentibacter salegens DSMZ5424 (Depositary institution; the Deutche Sammlung von Mikroorganismenund Zellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) which was seed cultured in this medium at 25° C.for 48 hours were applied on the same medium, followed by main culturingat 25° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 0.8 g of NH₄Cl, 0.25 g of KH₂PO₄, 0.4 g of K₂HPO₄, 0.505 g ofKNO₃, 15 mg of CaCl₂.2H₂O, 20 mg of MgCl₂.6H₂O, 7 mg of FeSO₄.7H₂O, 5 mgof Na₂SO₄, 5 mg of MnCl₂.4H₂O, 0.5 mg of H₃BO₃, 0.5 mg of ZnCl₂, 0.5 mgof CoCl₂.6H₂O, 0.5 mg of NiSO₄.6H₂O, 0.3 mg of CuCl₂.2H₂O, 10 mg ofNa₂MoO₄.2H₂O, 0.5 g of yeast extract, 0.5 g of peptone, 0.5 g ofcasamino acid, 0.5 g of dextrose, 0.5 g of soluble starch, 0.5 g ofsodium pyruvate, and 15 g of agar in 1 L of distilled water was used toculture Dyadobacter fermentans ATCC 700827 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America).Microbial cells of Dyadobacter fermentans ATCC 700827 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) which was seed cultured in this medium at 25° C. for 48 hours,followed by main culturing at 25° C. for 48 hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 2 g of tryptone, 0.5 g of meat extract, 0.5 g of yeastextract, 0.2 g of sodium acetate and 15 g of agar in 1 L of Daigoartificial sea water SP was used to culture Flammeovirga aprica NBRC15941 (Depositary institution; the NITE Biological Resource Center ofthe National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan). Microbial cells of Flammeovirga aprica NBRC 15941(Depositary institution; the NITE Biological Resource Center of theNational Institute of Technology and Evaluation, address of depositaryinstitution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken,Japan) which was seed cultured in this medium at 25° C. for 48 hourswere applied on the same medium, followed by main culturing at 25° C.for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 1 g of glucose, 1 g of peptone, 1 g of yeast extract, and 15g of agar in 1 L of distilled water was used to culture Spirosomalinguale DSMZ 74 (Depositary institution; the Deutche Sammlung vonMikroorganismen und Zellkulturen GmbH (German Collection of Microbes andCell Cultures, Address of Depositary institution; Mascheroder Weg 1b,38124 Braunschweig, Germany) or Flectobacillus major DSMZ 103(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany). Microbial cells of Spirosoma linguale DSMZ 74(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) or Flectobacillus major DSMZ 103 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany)which was seed cultured on this medium at 25° C. for 48 hours wereapplied on the same medium, followed by main culturing at 25° C. for 48hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 0.5 g of tryptone, 0.5 g of yeast extract, 0.2 g of meatextract, 0.2 g of sodium acetate and 15 g of agar in 300 ml of distilledwater and 700 ml of Daigo artificial sea water SP was used to cultureTenacibaculum maritimum ATCC 43398 (Depositary institution; the AmericanType Culture Collection, address of depositary institution; P.O. Box1549, Manassas, Va. 20110, the United States of America). Microbialcells of Tenacibaculum maritimum ATCC 43398 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America) whichwas seed cultured in this medium at 25° C. for 48 hours, followed bymain culturing at 25° C. for 48 hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 2.5 g of yeast extract, 2.5 g of tryptone, 100 mg ofnitrilotriacetic acid, 40 mg of CaSO₄.2H₂O, 200 mg of MgCl₂.6H₂O, 0.5 mlof 0.01M Fe citrate, 0.5 ml of a trace element solution, 100 ml ofphosphate buffer, 900 ml of distilled water, and 28 g of agar in 1 L wasused to culture Rhodothermus marinus DSMZ 4252 (Depositary institution;the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (GermanCollection of Microbes and Cell Cultures, Address of Depositaryinstitution; Mascheroder Weg 1b, 38124 Braunschweig, Germany). Note thatthe trace element solution contained 12.8 g of nitrilotriacetic acid, 1g of FeCl₂.4H₂O, 0.5 g of MnCl₂.4H₂O, 0.3 g of CoCl₂.4H₂O, 50 mg ofCuCl₂.2H₂O, 50 mg of Na₂MoO₄.2H₂O, 20 mg of H₃BO₃ and 20 mg ofNiCl₂.6H₂O). Microbial cells of Rhodothermus marinus DSMZ 4252(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) which was seed cultured in this medium at 60° C.for 48 hours were applied on the same medium, followed by main culturingat 60° C. for 48 hours.

An agar solid medium (1.5% agar, pH 7.6, sterilized at 120° C. for 15minutes) containing BACTO MARINE BROTH (DIFCO 2216) was used to cultureZobellia galactanivorans DSMZ 12802 (Depositary institution; the DeutcheSammlung von Mikroorganismen und Zellkulturen GmbH (German Collection ofMicrobes and Cell Cultures, Address of Depositary institution;Mascheroder Weg 1b, 38124 Braunschweig, Germany). This medium wasapplied with microbial cells of Zobellia galactanivorans DSMZ 12802(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) which was seed cultured in this medium at 30° C.for 48 hours were applied on the same medium, followed by main culturingat 30° C. for 48 hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 1.5 g of yeast extract, 2.5 g of peptone, 2 g of hexadecane,17.7 g of NaCl, 0.48 g of KCl, 3.4 g of MgCl₂.6H₂O, 4.46 g ofMgSO₄.7H₂O, 0.98 g of CaCl₂ and 15 g of agar in 1 L of distilled waterwas used to culture Muricauda ruestringenesis DSMZ 13258 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig,Germany). Microbial cells of Muricauda ruestringenesis DSMZ 13258(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) which was seed cultured in this medium at 30° C.for 48 hours were applied on the same medium, followed by main culturingat 30° C. for 48 hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 3 g of casitone, 1 g of yeast extract, 1.36 g of CaCl₂.2H₂Oand 15 g of agar in 1 L of distilled water was used to cultureTaxeobacter gelupurpurascens DSMZ 11116 (Depositary institution; theDeutche Sammlung von Mikroorganismen und Zellkulturen GmbH (GermanCollection of Microbes and Cell Cultures, Address of Depositaryinstitution; Mascheroder Weg 1b, 38124 Braunschweig, Germany). Microbialcells of Taxeobacter gelupurpurascens DSMZ 11116 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany)which was seed cultured in this medium at 30° C. for 48 hours wereapplied on the same medium, followed by main culturing at 30° C. for 48hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 3 g of casitone, 1 g of yeast extract, 1.36 g of CaCl₂.2H₂O,5 g of cellobiose and 15 g of agar in 1 L of distilled water was used toculture Cytophaga hutchinsonii NBRC 15051 (Depositary institution; theNITE Biological Resource Center of the National Institute of Technologyand Evaluation, address of depositary institution; 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan). Microbial cells of Cytophagahutchinsonii NBRC 15051 (Depositary institution; the NITE BiologicalResource Center of the National Institute of Technology and Evaluation,address of depositary institution; 5-8 Kazusa-Kamaashi 2-Chome,Kisarazu-shi, Chiba-ken, Japan) which was seed cultured in this mediumat 30° C. for 48 hours were applied on the same medium, followed by mainculturing at 30° C. for 48 hours.

An agar solid medium (pH 7.2, sterilized at 120° C. for 15 minutes)containing 10 g of peptone, 2 g of yeast extract, 0.5 g of MgSO₄.7H₂O,and 15 g of agar in 250 ml of distilled water and 750 ml of Daigoartificial sea water SP was used to culture Marinilabilia salmonicolorNBRC 15948 (Depositary institution; the NITE Biological Resource Centerof the National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan). Microbial cells of Marinilabilia salmonicolor NBRC15948 (Depositary institution; the NITE Biological Resource Center ofthe National Institute of Technology and Evaluation, address ofdepositary institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi,Chiba-ken, Japan) which was seed cultured in this medium at 30° C. for48 hours were applied on the same medium, followed by main culturing at30° C. for 48 hours.

An agar solid medium (pH 7.0, sterilized at 120° C. for 15 minutes)containing 0.5 g of KNO₃, 0.1 g of sodium glycerophosphate, 1 g oftrishydroxymethylaminomethane, 2 g of tryptone, 2 g of yeast extract, 15g of agar and 1 ml of a trace element solution in 1 L of Daigoartificial sea water SP was used to culture Saprospira grandis ATCC23119 ((Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America). Note that the trace element solutioncontained 2.85 g of H₃BO₄, 1.8 g of MnCl₂.4H₂O, 1.36 g of FeSO₄.7H₂O,26.9 mg of CuCl₂.2H₂O, 20.8 mg of ZnCl₂, 40.4 mg of CoCl₂.6H₂O, 25.2 mgof Na₂MoO₄.2H₂O and 1.77 g of sodium tartrate). Microbial cells ofSaprospira grandis ATCC 23119 (Depositary institution; the American TypeCulture Collection, address of depositary institution; P.O. Box 1549,Manassas, Va. 20110, the United States of America) which was seedcultured in this medium at 30° C. for 48 hours were applied on the samemedium, followed by main culturing at 30° C. for 48 hours.

An agar solid medium (pH 7.5, sterilized at 120° C. for 15 minutes)containing 27 mg of KH₂PO₄, 40 mg of K₂HPO₄, 40 mg of Na₂HPO₄.2H₂O, 50mg of CaCl₂.2H₂O, 75 mg of MgSO₄.7H₂O, 5 mg of FeCl₃.6H₂O, 3 mg ofMnSO₄.H₂O, 1.31 g of glutamic acid, 2.5 mg of Trypticase Soy Brothwithout glucose, 0.4 mg of thiamine, 0.01 mg of vitamin B12, 2 g ofglucose, and 1 ml of a trace element solution in 1 L of distilled waterwas used to culture Haliscomenobacter hydrossis ATCC 27775 ((Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica). Note that the trace element solution contained 0.1 g ofZnSO₄.7H₂O, 0.03 g of MnCl₂.4H₂O, 0.3 g of H₃BO₃, 0.2 g of CoCl₂.6H₂O,0.01 g of CuCl₂.2H₂O, 0.02 g of NiCl₂.6H₂O and 0.03 g of Na₂MoO₄.H₂O).Microbial cells of Haliscomenobacter hydrossis ATCC 27775 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) which was seed cultured in this medium at 25° C. for 48 hourswere applied on the same medium, followed by main culturing at 25° C.for 48 hours.

The thus obtained microbial cells were each collected from the agarmedium, and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mMEDTA to 100 g/L as wet microbial cells.

To 0.1 mL each of the microbial cell suspensions of these microbes wasadded 0.1 mL of 100 mM borate buffer (pH 9.0) containing 10 mM EDTA, 100mM L-aspartic acid-α,β-dimethyl ester hydrochloride and 200 mML-phenylalanine to make the total amount 0.2 mL. Then, reaction wascarried out at 20° C. for 3 hours when the microbe shown in Table 1-1was used or for 1 hour when the microbe shown in Table 1-2 was used. Theamount (mM) of α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP)produced is shown in Tables 1-1 and 1-2. Note that no β-AMP was detectedin all the cases where the microbes were used.

TABLE 1-1 α-AMP Microbe (mM) Aeromonas hydrophila ATCC 13136 1.55Azotobacter vinelandii IFO 3741 0.15 Alcaligenes faecalis FERM P-84600.37 Brevibacterium minutiferuna FERM BP-8277 0.10 Corynebacteriumflavescens ATCC 10340 0.26 Escherichia coli FERM BP-8276 3.68Empedobacter brevis ATCC 14234 6.31 Flavobacterium resinovorum ATCC14231 0.62 Microbacterium arborescens ATCC 4348 0.08 Propionibacteriumshermanii BERM BP-8100 3.41 Brevibacillus parabrevis ATCC 8185 0.08Paenibacillus alvei IFO 14175 0.09 Pseudomonas fragi IFO 3458 0.84Serratia grimesii ATCC 14460 0.47 Stenotrophomonas maltophilia ATCC13270 0.18 Sphingobacterium sp. FERM BP-8124 5.97 Streptomyceslavendulae NRRL B-1305 0.89 Xanthomonas maltophilia FERM BP-5568 0.40Williopsis saturnus IFO 0895 0.05 Candida magnoliae IFO 0705 0.26Geotrichum amycelium CBS 152.25 0.19 Geotrichum amycelium IFO 0905 0.06Saccharomyces unisporus IFO 0724 0.07 Torulaspora delbrueckii IFO 04220.04 Pichia ciferrii IFO 0905 0.06

TABLE 1-2 α-AMP α-AMP Microbe (mM) Microbe (mM) Cellulophaga lytica trSpirosoma linguale 0.15 NBRC 14961 DSMZ 74 Weeksella virosa trFlectobacillus major 0.68 NBRC 16016 DSMZ 103 Pedobacter heparinus 0.07Tenacibaculum maritimum tr NBRC 12017 ATCC 43398 Persicobacter diffluenstr Rhodotermus marinus 0.06 NBRC 15940 DSMZ 4252 Flexithrix dorotheae2.47 Zobellia galactanivorans 0.42 NBRC 15987 DSMZ 12802 Chitinophagapinensis 0.08 Muricauda ruestringensis 0.51 NBRC 15968 DSMZ 13258Cyclobacterium marinum 0.91 Salegentibacter salegens tr ATCC 25205 DSMZ5424 Runella slithyformis 0.07 Taxeobacter 0.02 ATCC 29530gelupurpurascens Thermonema lapsum tr DSMZ 11116 ATCC 43542 Cytophagahutchinsonii tr Psychroserpens 0.09 NBRC 15051 burtonensis Marinilabiliasalmonicolor 0.02 ATCC 700359 NBRC 15948 Gelidibacter algens 0.07Lewinella cohaerens 0.33 ATCC 700364 ATCC 23123 Dyadobacter fermentans0.04 Saprospira grandis 0.03 ATCC 700827 ATCC 23119 Flammeovirga aprica0.08 Haliscomenobacter tr NBRC 15941 hydrossis ATCC 27775

Reference Example 1 Microbe that Producesβ-L-aspartyl-L-phenyl-alanine-α-methyl ester

Microbes shown in Table 2 were cultured similarly to the procedure inbacteria in Table 1 of Example 1. After completion of the culturing, themicrobial cells were separated from these culture broths bycentrifugation, and suspended in 0.1 M borate buffer (pH 9.0) containing10 mM EDTA to 100 g/L as wet microbial cells. To 0.1 mL each of themicrobial cell suspensions of these microbes was added 0.1 mL of 100 mMborate buffer (pH 9.0) containing 10 mM EDTA, 100 mM L-asparticacid-α,β-dimethyl ester hydrochloride and 200 mM L-phenylalanine to makethe total amount 0.2 ml, followed by reaction at 30° C. for 2 hours. Theamount (mM) of β-L-aspartyl-L-phenylalanine-α-methyl ester (β-AMP)produced in this case is indicated in Table 2. Note that no α-AMP wasdetected in all the microbes.

TABLE 2 Microbe β-AMP (mM) Hafnia alvei ATCC 9760 0.30 Klebsiellapneumoniae ATCC 8308 0.26

Example 2 Purification of Enzyme from Empedobacter brevis

A 50 mL medium (pH 6.2) containing 5 grams (g) of glucose, 5 g ofammonium sulfate, 1 g of monopotassium phosphate, 3 g of dipotassiumphosphate, 0.5 g of magnesium sulfate, 10 g of yeast extract and 10 g ofpeptone in 1 liter (L) was transferred to a 500 mL Sakaguchi flask andsterilized at 115° C. for 15 minutes. This medium was then inoculatedwith 2 milliliters (ml or mL) of Empedobacter brevis strain FERM BP-8113(Depositary institution: the independent administrative corporation,National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Address of depositaryinstitution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan, International deposit transfer date: Jul. 8, 2002) that had beencultured at 30° C. for 16 hours in the same medium, followed by shakeculturing at 30° C. and 120 strokes/min for 16 hours.

Subsequently, the procedure after centrifugal separation was carried outeither on ice or at 4° C. The obtained culture broth was centrifuged tocollect microbial cells. After washing 16 g of the microbial cells with50 mM Tris-HCl buffer (pH 8.0), they were suspended in 40 milliliters(ml or mL) of the same buffer and subjected to ultrasonic crushingtreatment for 45 minutes at 195 watts. This ultrasonically crushedliquid was then centrifuged (10,000 rpm, 30 minutes) to remove thecrushed cell fragments and obtain an ultrasonic crushed liquidsupernatant. This ultrasonic crushed liquid supernatant was dialyzedovernight against 50 mM Tris-HCl buffer (pH 8.0) followed by removal ofthe insoluble fraction by ultracentrifugation (50,000 rpm, 30 minutes)to obtain a soluble fraction in the form of the supernatant liquid. Theresulting soluble fraction was applied to a Q-Sepharose HP column(manufactured by Amersham) pre-equilibrated with Tris-HCl buffer (pH8.0), and the active fraction was collected from the non-adsorbedfraction. This active fraction was dialyzed overnight against 50 mMacetate buffer (pH 4.5) followed by removal of the insoluble fraction bycentrifugal separation (10,000 rpm, 30 minutes) to obtain a dialyzedfraction in the form of the supernatant liquid. This dialyzed fractionwas then applied to a Mono S column (manufactured by Amersham)pre-equilibrated with 50 mM acetate buffer (pH 4.5) to elute enzyme at alinear concentration gradient of the same buffer containing 0 to 1 MNaCl. The fraction that had the lowest level of contaminating proteinamong the active fractions was applied to a Superdex 200 pg column(manufactured by Amersham) pre-equilibrated with 50 mM acetate buffer(pH 4.5) containing 1 M NaCl, and gel filtration was performed byallowing the same buffer (pH 4.5) containing 1 M NaCl to flow throughthe column to obtain an active fraction solution. As a result ofperforming these procedures, the peptide-forming enzyme used in thepresent invention was confirmed to have been uniformly purified based onthe experimental results of electrophoresis. The enzyme recovery rate inthe aforementioned purification process was 12.2% and the degree ofpurification was 707 times.

Example 3 Production of α-L-aspartyl-L-phenylalanine-β-methyl esterUsing Enzyme Fraction of Empedobacter brevis

10 microliters (μl) of Mono S fraction enzyme (about 20 U/ml) obtainedin Example 2 was added to 190 μl of borate buffer (pH 9.0) containing105.3 mM L-aspartic acid-α,β-dimethyl ester hydrochloride, 210.5 mML-phenylalanine and 10.51 mM EDTA and reaction was carried out at 20° C.The course of production of α-L-aspartyl-L-phenylalanine-β-methyl ester(α-AMP) is shown in Table 3. Note that almost no formation ofα-L-aspartyl-L-phenylalanine-β-methyl ester was confirmed in theenzyme-not-added lot.

Further, 10 μl of Mono S fraction enzyme (about 20 U/ml) obtained inExample 2 was added to 190 μl of borate buffer (pH 9.0) containing eachof 105.3 mM L-aspartic acid-α-methyl ester hydrochloride and L-asparticacid-β-methyl ester hydrochloride, 210.5 mM L-phenylalanine and 10.51 mMEDTA was added and reaction was carried out at 20° C. As a result, noformation of the corresponding peptides was observed.

TABLE 3 Reaction time (minute) Produced α-AMP (mM) 30 23.0 60 42.1 12061.7

Example 4 Purification of Enzyme from Sphingobacterium sp.

Sphingobacterium sp. strain FERM BP-8124 (Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit date:Jul. 22, 2002) was cultured in the same manner as that in Example 2using the medium shown in Example 2. The following procedure aftercentrifugal separation was carried out either on ice or at 4° C. Theobtained culture broth was centrifuged (10,000 rpm, 15 minutes) tocollect microbial cells. After washing 2 g of the microbial cells with20 mM Tris-HCl buffer (pH 7.6), they were suspended in 8 ml of the samebuffer and subjected to ultrasonic crushing treatment for 45 minutes at195 W. This ultrasonically crushed liquid was then centrifuged (10,000rpm, 30 minutes) to remove the crushed cell fragments to obtain anultrasonically crushed liquid supernatant. This ultrasonically crushedliquid supernatant was dialyzed overnight against 20 mM Tris-HCl buffer(pH 7.6) followed by removal of the insoluble fraction byultracentrifugation (50,000 rpm, 30 minutes) to obtain a solublefraction in the form of the supernatant liquid. The resulting solublefraction was applied to a Q-Sepharose HP column (manufactured byAmersham) pre-equilibrated with Tris-HCl buffer (pH 7.6), and the activefraction was collected from the non-adsorbed fraction. This activefraction was dialyzed overnight against 20 mM acetate buffer (pH 5.0)followed by removal of the insoluble fraction by centrifugal separation(10,000 rpm, 30 minutes) to obtain a dialyzed fraction in the form ofthe supernatant liquid. This dialyzed fraction was then applied to anSP-Sepharose HP column (manufactured by Amersham) pre-equilibrated with20 mM acetate buffer (pH 5.0) to obtain the active fraction in whichenzyme was eluted at a linear concentration gradient of the same buffercontaining 0 to 1 M NaCl.

Example 5 Production of α-L-aspartyl-L-phenylalanine-β-methyl ester andα-L-aspartyl-L-phenylalanine-β-ethyl ester Using Enzyme Fraction ofSphingobacterium sp.

In the case of production of α-L-aspartyl-L-phenylalanine-β-methyl ester(α-AMP), 15 μl of concentrated solution of SP-Sepharose HP fraction(about 15 U/ml) obtained in Example 4 was added to 185 μl of boratebuffer (pH 9.0) containing 108.1 mM L-aspartic acid-α,β-dimethyl esterhydrochloride, 216.2 mM L-phenylalanine and 10.8 mM EDTA and reactionwas carried out at 20° C. Similarly, in the case of production ofα-L-aspartyl-L-phenylalanine-β-ethyl ester (α-AEP), 10 μl of aconcentrated solution of SP-Sepharose HP fraction (about 15 U/ml)obtained in Example 4 was added to 190 μl of borate buffer (pH 9.0)containing 52.6 mM L-aspartic acid-α,β-diethyl ester hydrochloride,105.2 mM L-phenylalanine and 10.8 mM EDTA and reaction was carried outat 20° C. The course of formation of AMP or AEP is shown in Table 4.Note that almost no formation of AMP or AEP was confirmed in theenzyme-not-added lot. For formation of AEP, numerical values obtained byusing a standard product of AMP are described.

TABLE 4 Reaction time (minute) Produced α-AMP (mM) Produced α-AEP (mM)30 25.8 7.5 60 40.7 13.3 120 56.0 20.6 180 61.8 —

Example 6 Isolation of Peptide-forming Enzyme Gene Derived fromEmpedobacter brevis

Hereinafter, isolation of a peptide-forming enzyme gene will beexplained. Empedobacter brevis strain FERM BP-8113 (Depositaryinstitution: the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, Address of depositary institution: ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan,International deposit transfer date: Jul. 8, 2002) was used as themicrobe. In isolating the gene, Escherichia coli JM-109 was used as ahost while pUC118 was used as a vector.

(1) Production of PCR Primer Based on Determined Internal Amino AcidSequence

A mixed primer having the base sequences indicated in SEQ ID NO.: 3 andSEQ ID NO: 4, respectively, was produced based on the amino acidsequences (SEQ ID NOs: 1 and 2) determined according to the Edman'sdecomposition method from the digestion product of lysyl endopeptidaseof a peptide-forming enzyme derived from the Empedobacter brevis strainFERM BP-8113 (Depositary institution: the independent administrativecorporation, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, Address ofdepositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan, International deposit transfer date: Jul. 8, 2002).

(2) Acquisition of Microbial Cells

Empedobacter brevis strain FERM BP-8113 (Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit transferdate: Jul. 8, 2002) was cultured at 30° C. for 24 hours on a CM2G agarmedium (containing glucose at 50 g/l, yeast extract at 10 g/l, peptoneat 10 g/l, sodium chloride at 5 g/l, and agar at 20 g/l, pH 7.0). Oneloopful of the resulting microbial cells was inoculated into a 500 mlSakaguchi flask containing 50 ml of a CM2G liquid medium (theaforementioned medium excluding agar) followed by shake culturing at 30°C.

(3) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of culture broth was centrifuged (12,000 rpm, 4° C., 15 minutes)to collect the microbial cells. Then, a chromosomal DNA was acquiredfrom the microbial cells using the QIAGEN Genomic-Tip System (Qiagen)based on the procedure described in the manual therefor.

(4) Acquisition of DNA Fragment Containing Part of Peptide-formingEnzyme Gene by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Empedobacter brevis strain FERM BP-8113 (Depositaryinstitution: the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, Address of depositary institution: ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan,International deposit transfer date: Jul. 8, 2002) was acquired by thePCR method using LA-Taq (manufactured by Takara Shuzo). A PCR reactionwas then carried out on a chromosomal DNA acquired from Empedobacterbrevis strain FERM BP-8113 (Depositary institution: the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary,Address of depositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, Japan, International deposit transfer date:Jul. 8, 2002) using the primers having the base sequences of SEQ ID NOs:3 and 4.

The PCR reaction was carried out for 30 cycles under the followingconditions using the Takara PCR Thermal Cycler PERSONAL (manufactured byTakara Shuzo).

94° C. 30 seconds

52° C. 1 minute

72° C. 1 minute

After completion of the reaction, 3 μl of the reaction liquid wasapplied to 0.8% agarose electrophoresis. As a result, it was verifiedthat a DNA fragment of about 1.5 kilobases (kb) was amplified.

(5) Cloning of Peptide-Forming Enzyme Gene from Gene Library

In order to acquire the entire length of peptide-forming enzyme gene infull-length, Southern hybridization was carried out using the DNAfragment amplified in the PCR procedure as a probe. The procedure forSouthern hybridization is explained in Molecular Cloning, 2nd edition,Cold Spring Harbor Press (1989).

The approximately 1.5 kb DNA fragment amplified by the PCR procedure wasisolated by 0.8% agarose electrophoresis. The target band was then cutout and purified. This The DNA fragment was labeled with probedigoxinigen using DIG High Prime (manufactured by Boehringer-Mannheim)based on the procedure described in the manual therefor using DIG HighPrime (manufactured by Boehringer-Mannheim).

After completely digesting the chromosomal DNA of Empedobacter brevisacquired in the step (3) of the present Example 6 by reacting at 37° C.for 16 hours with restriction enzyme HindIII, the resultant waselectrophoresed with on 0.8% agarose gel. The electrophoresedchromosomal DNA was blotted onto a positively charged Nylon membranefilter (manufactured by Roche Diagnostics) from the agarose gel afterthe electrophoresis, followed by treatments consisting of alkalinedenaturation, neutralization and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 50° C. for 1 hour, the probe labeled withdigoxinigen prepared as described above was added and hybridization wascarried out at 50° C. for 16 hours. Subsequently, the filter was washedfor 20 minutes at room temperature with 2×SSC containing 0.1% SDS.Moreover, the filter was additionally washed twice at 65° C. for 15minutes with 0.1×SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim)based on the procedure described in the manual therefor using the DIGNucleotide Detection Kit (manufactured by Boehringer-Mannheim). As aresult, a roughly 4 kb band was able to be detected that hybridized withthe probe.

Then, 5 μg of the chromosomal DNA prepared in the step (3) of thepresent Example 6 was completely digested with HindIII. A roughly 4 kbof DNA was separated by 0.8% agarose gel electrophoresis, followed bypurification of the DNA using the Gene Clean II Kit (manufactured byFunakoshi) and dissolving the DNA in 10 μl of TE. 4 μl of this productwas then mixed with pUC118 HindIII/BAP (manufactured by Takara Shuzo)and a ligation reaction was carried out using the DNA Ligation Kit Ver.2 (manufactured by Takara Shuzo). 5 μl of the ligation reaction mixtureand 100 μl of competent cells of Escherichia coli JM109 (manufactured byToyobo) were mixed to transform the Escherichia coli. This was thenapplied to a suitable solid medium to produce a chromosomal DNA library.

To acquire the entire full-length of peptide-forming enzyme gene, thechromosomal DNA library was screened by colony hybridization using theaforementioned probe. The procedure for colony hybridization isexplained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter (Nylon Membrane for Colony and Plaque Hybridization,(manufactured by Roche Diagnostics) followed by treatments consisting ofalkali denaturation, neutralization and immobilization. Hybridizationwas carried out using EASY HYB (manufactured by Boehringer-Mannheim).After pre-hybridizing the filter at 37° C. for 1 hour, theaforementioned probe labeled with digoxinigen was added, followed byhybridization at 50° C. for 16 hours. Subsequently, the filter waswashed for 20 minutes at room temperature with 2×SSC containing 0.1%SDS. Moreover, the filter was additionally washed twice at 65° C. for 15minutes with 0.1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim). As a result, two strains of colonies were verifiedto hybridize with the labeled probe.

(6) Base Sequence of Peptide-Forming Enzyme Gene Derived fromEmpedobacter brevis

Plasmids possessed by Escherichia coli JM109 were prepared from theaforementioned two strains of microbial cells that were verified tohybridize with the labeled probe using the Wizard Plus Minipreps DNAPurification System (manufactured by Promega) to and the base sequenceof a portion where hybridization with the probe occurred and nearby wasdetermined. The sequencing reaction was carried out using the CEQDTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on theprocedure described in the manual therefor. In addition, electrophoresiswas carried out using the CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was verified that an open reading frame that encodes aprotein containing the internal amino acid sequences of thepeptide-forming enzyme (SEQ ID NOs: 1 and 2) did exist, therebyconfirming that the open reading frame was a gene encoding thepeptide-forming enzyme. The base sequence of the full-length of thepeptide-forming enzyme genes along with the corresponding amino acidsequences is shown in SEQ ID NO: 5 of the Sequence Listing. As a resultof analysis on the homology of the resulting open reading frame with theBLASTP program, homology was discovered between the two enzymes; itshowed with a homology of 34% as at the amino acid sequence levelexhibited with the α-amino acid ester hydrolase of Acetobacterpasteurianus (see Appl. Environ. Microbiol., 68(1), 211-218 (2002), anda homology of 26% at the amino acid sequence level exhibited with theglutaryl-7ACA acylase of Brevibacillus laterosporum (see J. Bacteriol.,173(24), 7848-7855 (1991).

(7) Expression of Peptide-forming Enzyme Gene Derived from Empedobacterbrevis in Escherichia coli

A target gene region on the promoter region of the trp operon on thechromosomal DNA of Escherichia coli W3110 was amplified by carrying outPCR using the oligonucleotides indicated in SEQ ID NOs: 7 and 8 asprimers, and the resulting DNA fragments were ligated to a pGEM-Teasyvector (manufactured by Promega). E. coli JM109 was then transformed inthis ligation solution, and those strains having the target plasmid inwhich the direction of the inserted trp promoter is inserted in theopposite to the orientation from of the lac promoter were selected fromampicillin-resistant strains. Next, a DNA fragment containing the trppromoter obtained by treating this plasmid with EcoO109I/EcoRI wasligated to an EcoO109I/EcoRI treatment product of pUC19 (manufactured byTakara). Escherichia coli JM109 was then transformed with this ligationsolution and those strains having the target plasmid were selected fromampicillin-resistant strains. Next, a DNA fragment obtained by treatingthis plasmid with HindIII/PvuII was ligated with to a DNA fragmentcontaining an rrnB terminator obtained by treating pKK223-3(manufactured by Amersham Pharmacia) with HindIII/HincII. E. coli JM109was then transformed with this ligation solution, strains having thetarget plasmid were selected from ampicillin-resistant strains, and theplasmid was designated as pTrpT.

The target gene was amplified by PCR using the chromosomal DNA ofEmpedobacter brevis strain FERM BP-8113 (Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit transferdate: Jul. 8, 2002) as a template and the oligonucleotides indicated inSEQ ID NO: 9 and 10 as primers. This DNA fragment was then treated withNdeI/PstI, and the resulting DNA fragment was ligated with the NdeI/PstItreatment product of pTrpT. Escherichia coli JM109 was then transformedwith this ligation solution, those strains having the target plasmidwere selected from ampicillin-resistant strains, and this plasmid wasdesignated as pTrpT_Gtg2.

Escherichia coli JM109 having pTrpT_Gtg2 was seed cultured at 30° C. for24 hours in LB medium containing 100 mg/l of ampicillin. 1 ml of theresulting culture broth was seeded in a 500 ml Sakaguchi flaskcontaining 50 ml of a medium (D glucose at 2 g/l, yeast extract at 10g/l, casamino acids at 10 g/l, ammonium sulfate at 5 g/l, potassiumdihydrogen phosphate at 3 g/l, dipotassium hydrogen phosphate at 1 g/l,magnesium sulfate heptahydrate at 0.5 g/l, and ampicillin at 100 mg/l),followed by culturing at 25° C. for 24 hours. The culture broth had anα-L-aspartyl-phenylalanine-β-methyl ester forming activity of 0.11 U per1 ml of culture broth and it was verified that the cloned gene wasexpressed by E. coli. Furthermore, no activity was detected for atransformant in which only pTrpT had been introduced as a control.

Prediction of Signal Sequence

When the amino acid sequence of SEQ ID NO: 6 described in the SequenceListing was analyzed with the Signal P v 1.1 program (see ProteinEngineering, Vol. 12, No. 1, pp. 3-9, 1999), it was predicted that aminoacids numbers 1 to 22 function as a signal that is secreted into theperiplasm, while the mature protein was estimated to be downstream ofamino acid number 23.

Verification of Secretion

Escherichia coli JM109, having pTrpT_Gtg2, was seed cultured at 30° C.for 24 hours in LB medium containing 100 mg/l of ampicillin. 1 ml of theresulting culture broth was seeded into a 500 ml Sakaguchi flaskcontaining 50 ml of medium (glucose at 2 g/l, yeast extract at 10 g/l,casamino acids at 10 g/l, ammonium sulfate at 5 g/l, potassiumdihydrogen phosphate at 3 g/l, dipotassium hydrogen phosphate at 1 g/l,magnesium sulfate heptahydrate at 0.5 g/l, and ampicillin at 100 mg/l),followed by final culturing at 25° C. for 24 hours to obtain culturedmicrobial cells.

The cultured microbial cells were fractionated into a periplasm fractionand a cytoplasm fraction by an osmotic pressure shock method using a 20grams/deciliter (g/dl) sucrose solution. The microbial cells immersed inthe 20 g/dl sucrose solution were immersed in a 5 mM aqueous MgSO₄solution. The centrifuged supernatant was named a periplasm fraction(“Pe”). In addition, the centrifuged sediment was re-suspended andsubjected to ultrasonic crushing. The resultant was named a cytoplasmfraction (“Cy”). The activity of glucose 6-phosphate dehydrogenase,which is known to be present in the cytoplasm, was used as an indicatorto verify that the cytoplasm had been separated. This measurement wascarried out by adding a suitable amount of enzyme to a reaction solutionat 30° C. containing 1 mM glucose 6-phosphate, 0.4 mM NADP, 10 mM MgSO₄,and 50 mM Tris-Cl (pH 8), followed by measurement of absorbance at 340nm to measure production of NADPH.

The amounts of enzymes of in the periplasm fraction and the cytoplasmfraction when the activity of a separately prepared cell-free extractwas assigned a value of 100% are shown in FIG. 1. That glucose6-phosphate dehydrogenase activity did not mix in the periplasm fractionindicates that the periplasm fraction did not mix in the cytoplasmfraction. About 60% of the α-L-aspartyl-L-phenylalanine-β-methyl ester(α-AMP) forming activity was recovered in the periplasm fraction, and itwas verified that the Ala-Gln-forming enzyme was secreted into theperiplasm as predicted from the amino acid sequence using the Signal P v1.1 program.

Example 7 Isolation of Peptide-forming Enzyme Gene Derived fromSphingobacterium sp.

Hereinafter, isolation of a peptide-forming enzyme gene is described.The microbe used was Sphingobacterium sp. strain FERM BP-8124(Depositary institution: the independent administrative corporation,National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Address of depositaryinstitution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan, International deposit date: Jul. 22, 2002). For the isolation ofthe gene, Escherichia coli DH5α was used as a host, and pUC118 was usedas a vector.

(1) Acquisition of Microbial Cells

Sphingobacterium sp. strain FERM BP-8124 (Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit date:Jul. 22, 2002) was cultured for 24 hours at 25° C. on a CM2G agar medium(containing glucose at 50 g/l, yeast extract at 10 g/l, peptone at 10g/l, sodium chloride at 5 g/l, and agar at 20 g/l, pH 7.0). One loopfulof the resulting microbial cells was inoculated into a 500 ml Sakaguchiflask containing 50 ml of CM2G liquid medium (the aforementioned mediumexcluding agar) followed by shake culturing at 25° C.

(2) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of culture broth was centrifuged (12,000 rpm, 4° C., 15 minutes)to collect the microbial cells. A chromosomal DNA was then acquired fromthe microbial cells using the Qiagen Genomic-Tip System (Qiagen) basedon the procedure described in the manual therefor.

(3) Acquisition of Probe DNA Fragment by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Empedobacter brevis strain FERM BP-8113 (Depositaryinstitution: the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary, Address of depositary institution: ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan,International deposit transfer date: Jul. 8, 2002) was acquired by thePCR method using LA-Taq (manufactured by Takara Shuzo). A PCR reactionwas then carried out on the chromosomal DNA acquired from Empedobacterbrevis strain FERM BP-8113 (Depositary institution: the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary,Address of depositary institution: Chuo Dai-6, 1-1 Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, Japan, International deposit transfer date:Jul. 8, 2002) using primers having the base sequences of SEQ ID NOs: 3and 4.

The PCR reaction was carried out using the Takara PCR ThermalCycler—PERSONAL (Takara Shuzo) for 30 cycles under the followingconditions.

94° C. 30 seconds 52° C. 1 minute 72° C. 1 minute

After completion of the reaction, 3 μl of reaction liquid was applied to0.8% agarose electrophoresis. As a result, it was verified that a DNAfragment of about 1.5 kb was amplified.

(4) Cloning of Peptide-forming Enzyme Gene from Gene Library

In order to acquire the full-length peptide-forming enzyme gene,Southern hybridization was carried out using the DNA fragment amplifiedin the aforementioned PCR procedure as a probe. The operations ofSouthern hybridization are explained in Molecular Cloning, 2nd edition,Cold Spring Harbor Press (1989).

The approximately 1.5 kb DNA fragment amplified by the aforementionedPCR procedure was separated by 0.8% agarose electrophoresis. The targetband was then cut out and purified. This DNA fragment was labeled withprobe digoxinigen using DIG High Prime (manufactured byBoehringer-Mannheim) based on the procedure described in the manualtherefor.

After allowing the chromosomal DNA of Sphingobacterium sp. acquired inthe step (2) of the present Example 7 to react with restriction enzymeSacI at 37° C. for 16 hours to completely digest the DNA, the resultantwas electrophoresed on 0.8% agarose gel. From the agarose gel after theelectrophoresis, the electrophoresed chromosomal DNA was blotted onto apositively charged Nylon membrane filter (manufactured by RocheDiagnostics), followed by treatments consisting of alkali denaturation,neutralization, and immobilization. Hybridization was carried out usingEASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizingthe filter at 37° C. for 1 hour, the digoxinigen-labeled probe preparedas described above was added and hybridization was carried out at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on theprocedure described in the manual therefor. As a result, a roughly 3 kbband was successfully detected that hybridized with the probe.

5 μg of the chromosomal DNA prepared in the step (2) of the presentExample 7 was completely digested with SacI. About 3 kb of a DNA wasseparated by 0.8% agarose gel electrophoresis, the DNA was purifiedusing the Gene Clean II Kit (manufactured by Funakoshi), and dissolvedin 10 μl of TE. 4 μl of the resulting solution and pUC118 treated withalkaline phosphatase (E. coli C75) at 37° C. for 30 minutes and at 50°C. for 30 minutes, after reaction with SacI at 37° C. for 16 hours tocompletely digest, were mixed and a ligation reaction was carried outusing the DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μlof this ligation reaction liquid and 100 μl of competent cells ofEscherichia coli DH5α (manufactured by Takara Shuzo) were mixed totransform the Escherichia coli. This was then applied to a suitablesolid medium to produce a chromosomal DNA library.

To acquire full-length peptide-forming enzyme gene, the chromosomal DNAlibrary was screened by colony hybridization using the aforementionedprobe. The procedure for colony hybridization is explained in MolecularCloning, 2nd edition, Cold Spring Harbor Press (1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter (Nylon Membrane for Colony and Plaque Hybridization,manufactured by Roche Diagnostics), followed by treatments of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 37° C. for 1 hour, the aforementioneddigoxinigen-labeled probe was added, followed by hybridization at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor. As a result, six strains of colonies were verified to havehybridized with the labeled probe.

(5) Base Sequence of Peptide-forming Enzyme Gene Derived fromSphingobacterium sp.

Plasmids possessed by Escherichia coli DH5α were prepared from the sixstrains of microbial cells that were verified to have hybridized withthe labeled probe using the Wizard Plus Minipreps DNA PurificationSystem (manufactured by Promega) to determine the base sequence of aportion where hybridization with the probe occurred and nearby wasdetermined. The sequencing reaction was carried out using the CEQDTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on theprocedure described in the manual therefor. In addition, electrophoresiswas carried out using the CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it revealed that an open reading frame that encodespeptide-forming enzyme did exist. The full-length base sequence of thepeptide-forming enzyme gene derived from Sphingobacterium sp. along withthe corresponding amino acid sequence is shown in SEQ ID NO: 11.Peptide-forming enzyme derived from Sphingobactenum sp. exhibited ahomology of 63.5% at the amino acid sequence level to thepeptide-forming enzyme derived from Empedobacter brevis (as determinedusing the BLASTP program).

(6) Expression of Peptide-forming Enzyme Gene Derived fromSphingobacterium sp. in Escherichia coli

The target gene was amplified by PCR using the chromosomal DNA ofSphingobacterium sp. FERM BP-8124 (Depositary institution: theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary, Address of depositary institution: Chuo Dai-6, 1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, International deposit date:Jul. 22, 2002) as a template and the oligonucleotides shown in SEQ IDNOs: 13 and 14 as primers. This DNA fragment was treated with NdeI/XbaI,and the resulting DNA fragment and an NdeI/XbaI treatment product ofpTrpT were ligated. Escherichia coli JM109 was then transformed withthis ligation solution, and strains having the target plasmid wereselected from ampicillin-resistant strains. The plasmid was designatedas pTrpT_Sm_aet.

Escherichia coli JM109 having pTrpT_Sm_aet was cultured at 25° C. for 20hours by inoculating one loopful thereof into an ordinary test tubecontaining 3 ml of a medium (glucose at 2 g/l, yeast extract at 10 g/l,casamino acids at 10 g/l, ammonium sulfate at 5 g/l, potassiumdihydrogen phosphate at 3 g/l, dipotassium hydrogen phosphate at 1 g/l,magnesium sulfate heptahydrate at 0.5 g/l and ampicillin at 100 mg/l).It was verified that a cloned gene having an α-AMP production activityof 0.53 U per ml of culture broth was expressed by Escherichia coli.Furthermore, no activity was detected for a transformant containing onlypTrpT used as a control.

Prediction of Signal Sequence

When the amino acid sequence of SEQ ID NO: 12 described in the SequenceListing was analyzed with the Signal P v 1.1 program (ProteinEngineering, Vol. 12, No. 1, pp. 3-9, 1999), it was predicted that aminoacids numbers 1 to 20 function as a signal that is secreted into theperiplasm, while the mature protein was estimated to be downstream ofamino acid number 21.

Confirmation of Signal Sequence

One loopful of Escherichia coli JM109, having pTrpT_Sm_aet, wasinoculated into ordinary test tubes containing 50 ml of a medium(glucose at 2 g/l, yeast extract at 10 g/l, casamino acids at 10 g/l,ammonium sulfate at 5 g/l, potassium dihydrogen phosphate at 3 g/l,dipotassium hydrogen phosphate at 1 g/l, magnesium sulfate heptahydrateat 0.5 g/l and ampicillin at 100 mg/l) and main culturing was performedat 25° C. for 20 hours.

Hereinafter, procedures after centrifugal separation were carried outeither on ice or at 4° C. After completion of the culturing, themicrobial cells were separated from the culture broth by centrifugation,washed with 100 mm phosphate buffer (pH 7), and then suspended in thesame buffer. The microbial cells were then subjected to ultrasoniccrushing treatment for 20 minutes at 195 W, the ultrasonic crushedliquid was centrifuged (12,000 rpm, 30 minutes) to remove the crushedcell fragments and obtain a soluble fraction. The resulting solublefraction was applied to a CHT-II column manufactured by Biorad)pre-equilibrated with 100 mM phosphate buffer (pH 7), and enzyme waseluted at a linear concentration gradient with 500 mM phosphate buffer.A solution obtained by mixing the active fraction with 5 time volumes of2 M ammonium sulfate and 100 mM phosphate buffer was applied to aResource-PHE column (manufactured by Amersham) pre-equilibrated with 2 Mammonium sulfate and 100 mM phosphate buffer, and an enzyme was elutedat a linear concentration gradient by 2 to 0 M ammonium sulfate toobtain an active fraction solution. As a result of these procedures, itwas verified that the peptide-forming enzyme was electrophoreticallyuniformly purified.

When the amino acid sequence of the aforementioned peptide-formingenzyme was determined by Edman's decomposition method, the amino acidsequence of SEQ ID NO: 15 was acquired, and the mature protein wasverified to be downstream of amino acid number 21 as was predicted bythe SignalP v 1.1 program.

Example 8 Isolation of Peptide-forming Enzyme Gene Derived fromPedobacter heparinus IFO 12017

Hereinafter, isolation of a peptide-forming enzyme gene is described.The microbe used is Pedobacter heparinus IFO 12017 (Depositaryinstitution; the Institute of Fermentation, Osaka, address of thedepositary institution; Feb. 17, 1985 Jusanbon-cho, Yodogawa-ku,Osaka-shi, Japan). For the isolation of the gene, Escherichia coli JM109was used as a host, and pUC118 was used as a vector.

(1) Acquisition of Microbial Cells

Pedobacter heparinus IFO 12017(Depositary institution: the Institute ofFermentation, Osaka; Feb. 17, 1985 Jusanbon-cho, Yodogawa-ku, Osaka-shi,Japan) was cultured for 24 hours at 25° C. on a CM2G agar medium(containing glucose at 50 g/l, yeast extract at 10 g/l, peptone at 10g/l, sodium chloride at 5 g/l, and agar at 20 g/l, pH 7.0). One loopfulof the resulting microbial cells was inoculated into a 500 ml Sakaguchiflask containing 50 ml of CM2G liquid medium (the aforementioned mediumexcluding agar) followed by shake culturing at 25° C.

(2) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of the culture broth was centrifuged (12,000 rpm, 4° C., 15minutes) to collect the microbial cells. A chromosomal DNA was thenacquired from the microbial cells using the Qiagen Genomic-Tip System(Qiagen) based on the procedure described in the manual therefor.

(3) Acquisition of Probe DNA Fragment by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Pedobacter heparinus IFO 12017(Depositary institution: theInstitute of Fermentation, Osaka; Feb. 17, 1985 Jusanbon-cho,Yodogawa-ku, Osaka-shi, Japan) was acquired by the PCR method usingLA-Taq (manufactured by Takara Shuzo). A PCR reaction was then carriedout on the chromosomal DNA acquired from Pedobacter heparinus IFO 12017(Depositary institution: the Institute of Fermentation, Osaka; Feb. 17,1985 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) using primers havingthe base sequences of SEQ ID NOs: 15 and 16. The approximately 1 kb DNAfragment amplified by the PCR procedure was isolated by 0.8% agaroseelectrophoresis. The target band was then cut out and purified. The DNAfragment was labeled with probe digoxinigen using DIG High Prime(manufactured by Boehringer-Mannheim) based on the procedure describedin the manual therefor.

(4) Cloning of Peptide-forming Enzyme Gene from Gene Library

To acquire the full-length peptide-forming enzyme gene, Southernhybridization was carried out using the DNA fragment amplified in theaforementioned PCR procedure as a probe. The operations of Southernhybridization are explained in Molecular Cloning, 2nd edition, ColdSpring Harbor Press (1989).

After allowing the chromosomal DNA of Pedobacter heparinus IFO 12017(Depositary institution: the Institute of Fermentation, Osaka; Feb. 17,1985 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) to react withrestriction enzyme HindIII at 37° C. for 16 hours to completely digestthe DNA, the resultant was electrophoresed on 0.8% agarose gel. From theagarose gel after the electrophoresis, the electrophoresed chromosomalDNA was blotted onto a positively charged Nylon membrane filter(manufactured by Roche Diagnostics), followed by treatments consistingof alkali denaturation, neutralization, and immobilization.Hybridization was carried out using EASY HYB (manufactured byBoehringer-Mannheim). After pre-hybridizing the filter at 50° C. for 1hour, the digoxinigen-labeled probe prepared as described above wasadded and hybridization was carried out at 50° C. for 16 hours.Subsequently, the filter was washed twice at 60° C. with 1×SSCcontaining 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on theprocedure described in the manual therefor. As a result, a roughly 5 kbband was successfully detected that hybridized with the probe.

5 μg of the chromosomal DNA of Pedobacter heparinus IFO 12017 wascompletely digested with HindIII. About 5 kb of a DNA was separated by0.8% agarose gel electrophoresis, the DNA was purified using the GeneClean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE.4 μl of the resulting solution and pUC118 HindIII/BAP were mixed weremixed and a ligation reaction was carried out using the DNA Ligation KitVer. 2 (manufactured by Takara Shuzo). 5 μl of this ligation reactionliquid and 100 μl of competent cells of Escherichia coli JM109(manufactured by Takara Shuzo) were mixed to transform the Escherichiacoli. This was then applied on a suitable solid medium to produce achromosomal DNA library.

To acquire a full-length peptide-forming enzyme gene, the chromosomalDNA library was screened by colony hybridization using theaforementioned probe. The procedure for colony hybridization isexplained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter, Nylon Membrane for Colony and Plaque Hybridization(manufactured by Roche Diagnostics), followed by treatments of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 37° C. for 1 hour, the aforementioneddigoxinigen-labeled probe was added, followed by hybridization at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor. As a result, one strain whose colony hybridized with thelabeled probe was observed.

(5) Base Sequence of Peptide-forming Enzyme Gene Derived from Pedobacterheparinus IFO 12017

Plasmids possessed by Escherichia coli JM109 were prepared from thestrain that was verified to have hybridized with the labeled probe andthe base sequence of a portion where hybridization with the probeoccurred and nearby was determined. The sequencing reaction was carriedout using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter)based on the procedure described in the manual therefor. In addition,electrophoresis was carried out using the CEQ 2000-XL (manufactured byBeckman-Coulter).

As a result, it revealed that an open reading frame that encodespeptide-forming enzyme did exist. The full-length base sequence of thepeptide-forming enzyme gene derived from Pedobacter heparinus IFO 12017(Depositary institution: the Institute of Fermentation, Osaka; Feb. 17,1985 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) along with thecorresponding amino acid sequence is shown in SEQ ID NO: 17.

Example 9 Expression of Peptide-forming Enzyme Gene Derived fromPedobacter heparinus IFO 12017 in Escherichia coli

The target gene was amplified by PCR using the chromosomal DNA ofPedobacter heparinus IFO 12017 (Depositary institution: the Institute ofFermentation, Osaka; Feb. 17, 1985 Jusanbon-cho, Yodogawa-ku, Osaka-shi,Japan) as a template and the oligonucleotides shown in SEQ ID NOs: 19and 20 as primers. This DNA fragment was treated with NdeI/HindIII, andthe resulting DNA fragment and an NdeI/HindIII treatment product ofpTrpT were ligated. Escherichia coli JM109 was then transformed withthis ligation solution, and strains having the target plasmid wereselected from ampicillin-resistant strains. The plasmid was designatedas pTrpT_Ph_aet.

One loopful of Escherichia coli JM109 having pTrpT_Ph_aet was inoculatedin an ordinary test tube containing 3 ml of a medium (glucose at 2 g/l,yeast extract at 10 g/l, casamino acids at 10 g/l, ammonium sulfate at 5g/l, potassium dihydrogen phosphate at 3 g/l, dipotassium hydrogenphosphate at 1 g/l, magnesium sulfate heptahydrate at 0.5 g/l andampicillin at 100 mg/l) and main culturing was performed at 25° C. for20 hours. It was verified that the cultured broth had an α-AMPproduction activity of 0.01 U per ml of culture broth so that it wasverified that the cloned gene was expressed in Escherichia coli.Furthermore, no activity was detected in the transformant containingonly pTrpT used as a control.

Example 10 Isolation of Peptide-forming Enzyme Gene Derived fromTaxeobacter gelupurpurascens DSMZ 11116

Hereinafter, isolation of a peptide-forming enzyme gene is described.The microbe used is Taxeobacter gelupurpurascens DSMZ 11116 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures); address of thedepositary institution; Mascheroder Weg 1b, 38124 Braunschweig,Germany). For the isolation of the gene, Escherichia coli JM109 was usedas a host, and pUC118 was used as a vector.

(1) Acquisition of Microbial Cells

Taxeobacter gelupurpurascens DSMZ 11116 (Depositary institution; theDeutche Sammlung von Mikroorganismen und Zellkulturen GmbH (GermanCollection of Microbes and Cell Cultures, Address of Depositaryinstitution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) wascultured for 24 hours at 25° C. on a CM2G agar medium (containingglucose at 50 g/l, yeast extract at 10 g/l, peptone at 10 g/l, sodiumchloride at 5 g/l, and agar at 20 g/l, pH 7.0). One loopful of theresulting microbial cells was inoculated into a 500 ml Sakaguchi flaskcontaining 50 ml of CM2G liquid medium (the aforementioned mediumexcluding agar) followed by shake culturing at 25° C.

(2) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of the culture broth was centrifuged (12,000 rpm, 4° C., 15minutes) to collect the microbial cells. A chromosomal DNA was thenacquired from the microbial cells using the Qiagen Genomic-Tip System(Qiagen) based on the procedure described in the manual therefor.

(3) Acquisition of Probe DNA Fragment by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Taxeobacter gelupurpurascens DSMZ 11116 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany)was acquired by the PCR method using LA-Taq (manufactured by TakaraShuzo). A PCR reaction was then carried out on the chromosomal DNAacquired from Taxeobacter gelupurpurascens DSMZ 11116 (Depositaryinstitution; the Deutche Sammlung von Mikroorganismen und ZellkulturenGmbH (German Collection of Microbes and Cell Cultures, Address ofDepositary institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany)using primers having the base sequences of SEQ ID NOs: 21 and 16. Theapproximately 1 kb DNA fragment amplified by the PCR procedure wasisolated by 0.8% agarose electrophoresis. The target band was then cutout and purified. The DNA fragment was labeled with probe digoxinigenusing DIG High Prime (manufactured by Boehringer-Mannheim) based on theprocedure described in the manual therefor.

(4) Cloning of Peptide-forming Enzyme Gene from Gene Library

To acquire the full-length peptide-forming enzyme gene, Southernhybridization was carried out using the DNA fragment amplified in theaforementioned PCR procedure as a probe. The operations of Southernhybridization are explained in Molecular Cloning, 2nd edition, ColdSpring Harbor Press (1989).

After allowing the chromosomal DNA of Taxeobacter gelupurpurascens DSMZ11116 (Depositary institution; the Deutche Sammlung von Mikroorganismenund Zellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) to react with restriction enzyme PstI at 37° C.for 16 hours to completely digest the DNA, the resultant waselectrophoresed on 0.8% agarose gel. From the agarose gel after theelectrophoresis, the electrophoresed chromosomal DNA was blotted onto apositively charged Nylon membrane filter (manufactured by RocheDiagnostics), followed by treatments consisting of alkali denaturation,neutralization, and immobilization. Hybridization was carried out usingEASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizingthe filter at 50° C. for 1 hour, the digoxinigen-labeled probe preparedas described above was added and hybridization was carried out at 50° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on theprocedure described in the manual therefor. As a result, a roughly 5 kbband was successfully detected that hybridized with the probe.

5 μg of the chromosomal DNA of Taxeobacter gelupurpurascens DSMZ 11116(Depositary institution; the Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microbes and Cell Cultures,Address of Depositary institution; Mascheroder Weg 1b, 38124Braunschweig, Germany) was completely digested with PstI. About 5 kb ofa DNA was separated by 0.8% agarose gel electrophoresis, the DNA waspurified using the Gene Clean II Kit (manufactured by Funakoshi), anddissolved in 10 μl of TE. 4 μl of the resulting solution and pUC118PstI/BAP (manufactured by Takara Shuzo) were mixed were mixed and aligation reaction was carried out using the DNA Ligation Kit Ver. 2(manufactured by Takara Shuzo). 5 μl of this ligation reaction liquidand 100 μl of competent cells of Escherichia coli JM109 (manufactured byTakara Shuzo) were mixed to transform the Escherichia coli. This wasthen applied on a suitable solid medium to produce a chromosomal DNAlibrary.

To acquire a full-length peptide-forming enzyme gene, the chromosomalDNA library was screened by colony hybridization using theaforementioned probe. The procedure for colony hybridization isexplained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter, Nylon Membrane for Colony and Plaque Hybridization(manufactured by Roche Diagnostics), followed by treatments of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 37° C. for 1 hour, the aforementioneddigoxinigen-labeled probe was added, followed by hybridization at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor. As a result, one strain whose colony hybridized with thelabeled probe was observed.

(5) Base Sequence of Peptide-forming Enzyme Gene Derived fromTaxeobacter gelupurpurascens DSMZ 11116

Plasmids possessed by Escherichia coli JM109 were prepared from thestrain that was verified to have hybridized with the labeled probe andthe base sequence of a portion where hybridization with the probeoccurred nearby was determined. The sequencing reaction was carried outusing the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter)based on the procedure described in the manual therefor. In addition,electrophoresis was carried out using the CEQ 2000-XL (manufactured byBeckman-Coulter).

As a result, it revealed that an open reading frame that encodespeptide-forming enzyme did exist. The full-length base sequence of thepeptide-forming enzyme gene derived from Taxeobacter gelupurpurascensDSMZ 11116 along with the corresponding amino acid sequence is shown inSEQ ID NO: 22.

Example 11 Isolation of Peptide-forming Enzyme Gene Derived fromCyclobacterium marinum ATCC 25205

Hereinafter, isolation of a peptide-forming enzyme gene is described.The microbe used is Cyclobacterium marinum ATCC 25205 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica). For the isolation of the gene, Escherichia coli JM109 was usedas a host, and pUC118 was used as a vector.

(1) Acquisition of Microbial Cells

Cyclobacterium marinum ATCC 25205 (Depositary institution; the AmericanType Culture Collection, address of depositary institution; P.O. Box1549, Manassas, Va. 20110, the United States of America) was culturedfor 24 hours at 25° C. on a CM2G agar medium (containing glucose at 50g/l, yeast extract at 10 g/l, peptone at 10 g/l, sodium chloride at 5g/l, and agar at 20 g/l, pH 7.0). One loopful of the resulting microbialcells was inoculated into a 500 ml Sakaguchi flask containing 50 ml ofCM2G liquid medium (the aforementioned medium excluding agar) followedby shake culturing at 25° C.

(2) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of the culture broth was centrifuged (12,000 rpm, 4° C., 15minutes) to collect the microbial cells. A chromosomal DNA was thenacquired from the microbial cells using the Qiagen Genomic-Tip System(Qiagen) based on the procedure described in the manual therefor.

(3) Acquisition of Probe DNA Fragment by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Cyclobacterium marinum ATCC 25205 was acquired by the PCRmethod using LA-Taq (manufactured by Takara Shuzo). A PCR reaction wasthen carried out on the chromosomal DNA acquired from Cyclobacteriummarinum ATCC 25205 (Depositary institution; the American Type CultureCollection, address of depositary institution; P.O. Box 1549, Manassas,Va. 20110, the United States of America) using primers having the basesequences of SEQ ID NOs: 15 and 16. The approximately 1 kb DNA fragmentamplified by the PCR procedure was isolated by 0.8% agaroseelectrophoresis. The target band was then cut out and purified. The DNAfragment was labeled with probe digoxinigen using DIG High Prime(manufactured by Boehringer-Mannheim) based on the procedure describedin the manual therefor.

(4) Cloning of Peptide-forming Enzyme Gene from Gene Library

To acquire the full-length peptide-forming enzyme gene, Southernhybridization was carried out using the DNA fragment amplified in theaforementioned PCR procedure as a probe. The operations of Southernhybridization are explained in Molecular Cloning, 2nd edition, ColdSpring Harbor Press (1989).

After allowing the chromosomal DNA of Cyclobacterium marinum ATCC 25205(Depositary institution; the American Type Culture Collection, addressof depositary institution; P.O. Box 1549, Manassas, Va. 20110, theUnited States of America) to react with restriction enzyme PstI orHincII at 37° C. for 16 hours to completely digest the DNA, theresultant was electrophoresed on 0.8% agarose gel. From the agarose gelafter the electrophoresis, the electrophoresed chromosomal DNA wasblotted onto a positively charged Nylon membrane filter (manufactured byRoche Diagnostics), followed by treatments consisting of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 50° C. for 1 hour, the digoxinigen-labeledprobe prepared as described above was added and hybridization wascarried out at 50° C. for 16 hours. Subsequently, the filter was washedtwice at 60° C. with 1×SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on theprocedure described in the manual therefor. As a result, a 7 kb bandthat hybridized with the probe was successfully detected for thePstI-digested product and a 2 kb band that hybridized with the probe wassuccessfully detected for the HincII-digested product.

5 μg of the chromosomal DNA of Cyclobacterium marinum ATCC 25205(Depositary institution; the American Type Culture Collection, addressof depositary institution; P.O. Box 1549, Manassas, Va. 20110, theUnited States of America) was completely digested with PstI or HincII.About 7 kb or 2 kb DNA was separated by 0.8% agarose gelelectrophoresis. The DNA was purified using the Gene Clean II Kit(manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of theresulting solution and pUC118 PstI/BAP (manufactured by Takara Shuzo) orpUC118 HincII/BAP (manufactured by Takara Shuzo) were mixed and aligation reaction was carried out using the DNA Ligation Kit Ver. 2(manufactured by Takara Shuzo). 5 μl of this ligation reaction liquidand 100 μl of competent cells of Escherichia coli JM109 (manufactured byTakara Shuzo) were mixed to transform the Escherichia coli. This wasthen applied on a suitable solid medium to produce a chromosomal DNAlibrary.

To acquire a full-length peptide-forming enzyme gene, the chromosomalDNA library was screened by colony hybridization using theaforementioned probe. The procedure for colony hybridization isexplained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter, Nylon Membrane for Colony and Plaque Hybridization(manufactured by Roche Diagnostics), followed by treatments of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 37° C. for 1 hour, the aforementioneddigoxinigen-labeled probe was added, followed by hybridization at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor. As a result, one strain each whose colony hybridized with thelabeled probe was observed.

(5) Base Sequence of Peptide-forming Enzyme Gene Derived fromCyclobacterium marinum ATCC 25205

Plasmids possessed by Escherichia coli JM109 were prepared from eachstrain that was verified to have hybridized with the labeled probe andthe base sequence of a portion where hybridization with the probeoccurred and nearby was determined. The sequencing reaction was carriedout using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter)based on the procedure described in the manual therefor. In addition,electrophoresis was carried out using the CEQ 2000-XL (manufactured byBeckman-Coulter).

As a result, it revealed that an open reading frame that encodespeptide-forming enzyme did exist. The full-length base sequence of thepeptide-forming enzyme gene derived from Cyclobacterium marinum ATCC25205(Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America) along with the corresponding amino acidsequence is shown in SEQ ID NO: 24.

Example 12 Isolation of Peptide-forming Enzyme Gene Derived fromPsychroserpens burtonensis ATCC 700359

Hereinafter, isolation of a peptide-forming enzyme gene is described.The microbe used is Psychroserpens burtonensis ATCC 700359 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica). For the isolation of the gene, Escherichia coli JM109 was usedas a host, and pUC118 was used as a vector.

(1) Acquisition of Microbial Cells

Psychroserpens burtonensis ATCC 700359 (Depositary institution; theAmerican Type Culture Collection, address of depositary institution;P.O. Box 1549, Manassas, Va. 20110, the United States of America) wascultured for 24 hours at 25° C. on a CM2G agar medium (containingglucose at 50 g/l, yeast extract at 10 g/l, peptone at 10 g/l, sodiumchloride at 5 g/l, and agar at 20 g/l, pH 7.0). One loopful of theresulting microbial cells was inoculated into a 500 ml Sakaguchi flaskcontaining 50 ml of CM2G liquid medium (the aforementioned mediumexcluding agar) followed by shake culturing at 10° C.

(2) Acquisition of Chromosomal DNA from Microbial Cells

50 ml of the culture broth was centrifuged (12,000 rpm, 4° C., 15minutes) to collect the microbial cells. A chromosomal DNA was thenacquired from the microbial cells using the Qiagen Genomic-Tip System(Qiagen) based on the procedure described in the manual therefor.

(3) Acquisition of Probe DNA Fragment by PCR

A DNA fragment containing a portion of the peptide-forming enzyme genederived from Psychroserpens burtonensis ATCC 700359 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) was acquired by the PCR method using LA-Taq (manufactured byTakara Shuzo). A PCR reaction was then carried out on the chromosomalDNA acquired from Psychroserpens burtonensis ATCC 700359 (Depositaryinstitution; the American Type Culture Collection, address of depositaryinstitution; P.O. Box 1549, Manassas, Va. 20110, the United States ofAmerica) using primers having the base sequences of SEQ ID NOs: 15 and16. The approximately 1 kb. DNA fragment amplified by the PCR procedurewas isolated by 0.8% agarose electrophoresis. The target band was thencut out and purified. The DNA fragment was labeled with probedigoxinigen using DIG High Prime (manufactured by Boehringer-Mannheim)based on the procedure described in the manual therefor.

(4) Cloning of Peptide-forming Enzyme Gene from Gene Library

To acquire the full-length peptide-forming enzyme gene, Southernhybridization was carried out using the DNA fragment amplified in theaforementioned PCR procedure as a probe. The operations of Southernhybridization are explained in Molecular Cloning, 2nd edition, ColdSpring Harbor Press (1989).

After allowing the chromosomal DNA of Psychroserpens burtonensis ATCC700359 to react with restriction enzyme EcoRI at 37° C. for 16 hours tocompletely digest the DNA, the resultant was electrophoresed on 0.8%agarose gel. From the agarose gel after the electrophoresis, theelectrophoresed chromosomal DNA was blotted onto a positively chargedNylon membrane filter (manufactured by Roche Diagnostics), followed bytreatments consisting of alkali denaturation, neutralization, andimmobilization. Hybridization was carried out using EASY HYB(manufactured by Boehringer-Mannheim). After pre-hybridizing the filterat 50° C. for 1 hour, the digoxinigen-labeled probe prepared asdescribed above was added and hybridization was carried out at 50° C.for 16 hours. Subsequently, the filter,was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was carried out usingthe DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on theprocedure described in the manual therefor. As a result, a roughly 7 kbband was successfully detected that hybridized with the probe.

5 μg of the chromosomal DNA of Psychroserpens burtonensis ATCC 700359was completely digested with EcoRI. About 7 kb of a DNA was separated by0.8% agarose gel electrophoresis, the DNA was purified using the GeneClean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE.4 μl of the resulting solution and pUC118 EcoRI/BAP (manufactured byTakara Shuzo) were mixed were mixed and a ligation reaction was carriedout using the DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5μl of this ligation reaction liquid and 100 μl of competent cells ofEscherichia coli JM109 (manufactured by Takara Shuzo) were mixed totransform the Escherichia coli. This was then applied on a suitablesolid medium to produce a chromosomal DNA library.

To acquire a full-length peptide-forming enzyme gene, the chromosomalDNA library was screened by colony hybridization using theaforementioned probe. The procedure for colony hybridization isexplained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

The colonies of the chromosomal DNA library were transferred to a Nylonmembrane filter, Nylon Membrane for Colony and Plaque Hybridization(manufactured by Roche Diagnostics), followed by treatments of alkalidenaturation, neutralization, and immobilization. Hybridization wascarried out using EASY HYB (manufactured by Boehringer-Mannheim). Afterpre-hybridizing the filter at 37° C. for 1 hour, the aforementioneddigoxinigen-labeled probe was added, followed by hybridization at 37° C.for 16 hours. Subsequently, the filter was washed twice at 60° C. with1×SSC containing 0.1% SDS.

Detection of colonies that hybridized with the labeled probe was carriedout using the DIG Nucleotide Detection Kit (manufactured byBoehringer-Mannheim) based on the explanation described in the manualtherefor. As a result, one strain whose colony hybridized with thelabeled probe was observed.

(5) Base Sequence of Peptide-forming Enzyme Gene Derived fromPsychroserpens burtonensis ATCC 700359

Plasmids possessed by Escherichia coli JM109 were prepared from thestrain that was verified to have hybridized with the labeled probe andthe base sequence of a portion where hybridization with the probeoccurred and nearby was determined. The sequencing reaction was carriedout using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter)based on the procedure described in the manual therefor. In addition,electrophoresis was carried out using the CEQ 2000-XL (manufactured byBeckman-Coulter).

As a result, it revealed that an open reading frame that encodespeptide-forming enzyme did exist. The full-length base sequence of thepeptide-forming enzyme gene derived from Psychroserpens burtonensis ATCC700359(Depositary institution; the American Type Culture Collection,address of depositary institution; P.O. Box 1549, Manassas, Va. 20110,the United States of America) along with the corresponding amino acidsequence is shown in SEQ ID NO: 26.

Although the invention has been described with respect to a specificembodiment for a complete and clear disclosure, the appended claims arenot to be thus limited but are to be construed as embodying allmodifications and alternative constructions that may occur to oneskilled in the art which fairly fall within the basic teaching hereinset forth.

Sequence Listing Free Text

-   SEQ ID NO: 3: Synthetic primer 1-   SEQ ID NO: 4: Synthetic primer 2-   SEQ ID NO: 5: Gene encoding a peptide-forming enzyme-   SEQ ID NO: 7: Synthetic primer for preparing pTrpT-   SEQ ID NO: 8: Synthetic primer for preparing pTrpT-   SEQ ID NO: 9: Synthetic primer for preparing pTrpT_Gtg2-   SEQ ID NO: 10: Synthetic primer for preparing pTrpT_Gtg2-   SEQ ID NO: 11: Gene encoding a peptide-forming enzyme-   SEQ ID NO: 13: Synthetic primer for preparing pTrpT_Sm_aet-   SEQ ID NO: 14: Synthetic primer for preparing pTrpT_Sm_aet-   SEQ ID NO: 15: Mix primer 1 for Aet-   SEQ ID NO: 16: Mix primer 2 for Aet-   SEQ ID NO: 19: Primer 1 for constructing aet expression vectors    derived from pPedobacter-   SEQ ID NO: 20: Primer 2 for constructing aet expression vectors    derived from pedobacterPedobacter-   SEQ ID NO: 21: Mix primer 3 for Aet

1. A method of producing an α-L-aspartyl-L-phenylalanine-β-ester,comprising forming the α-L-aspartyl-L-phenylalanine-β-ester fromL-aspartic acid-α,β-diester and L-phenylalanine using an enzyme orenzyme-containing substance that has an ability to selectively linkL-phenylalanine to an α-ester site of the L-aspartic acid-α,β-diesterthrough a peptide bond, wherein said enzyme or an enzyme in saidenzyme-containing substance is selected from the group consisting of: aprotein having the amino acid sequence consisting of amino acid residue21 to 619 of SEQ ID NO: 12, a protein having an amino acid sequenceincluding substitution, deletion, insertion, and/or addition of one tothirty amino acids in the amino acid sequence consisting of amino acidresidues 21 to 619 of SEQ ID NO: 12, and having activity to selectivelylink L-phenylalanine to an α-ester site of the L-asparticacid-α,β-diester through a peptide bond, a protein having the amino acidsequence of SEQ ID NO: 12, and a protein containing a mature proteinregion, having an amino acid sequence including substitution, deletion,insertion, and/or addition of one to thirty amino acids in the aminoacid sequence of SEQ ID NO: 12, and having activity to selectively linkL-phenylalanine to an α-ester site of the L-aspartic acid-α,β-diesterthrough a peptide bond.
 2. The method for producing anα-L-aspartyl-L-phenylalanine-β-ester according to claim 1, wherein theenzyme or enzyme-containing substance is one type or two or more typesselected from the group consisting of a culture of a microbe that has anability to selectively link L-phenylalanine to an α-ester site of theL-aspartic acid-α,β-diester through a peptide bond, a microbial cellseparated from the culture and a treated microbial cell product of themicrobe.
 3. The method for producing anα-L-aspartyl-L-phenylalanine-β-ester according to claim 2, wherein themicrobe is a microbe belonging to a genus selected from the groupconsisting of Aeromonas, Azotobacter, Alcaligenes, Brevibacterium,Corynebacterium, Escherichia, Empedobacter, Flavobacterium,Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus,Pseudomonas, Serratia, Stenotrophomonas, Sphingobacterium, Streptomyces,Xanthomonas, Williopsis, Candida, Geotrichum, Pichia, Saccharomyces,Torulaspora, Cellulophaga, Weeksella, Pedobacter, Persicobacter,Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema,Psychroserpens, Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma,Flectobacillus, Tenacibaculum, Rhodotermus, Zobellia, Muricauda,Salegentibacter, Taxeobacter, Cytophaga, Marinilabilia, Lewinella,Saprospira, and Haliscomenobacter.
 4. The method for producing anα-L-aspartyl-L-phenylalanine-β-ester according to claim 2, wherein themicrobe is a transformed microbe that is capable of expressing a proteinselected from the group consisting of: a protein having an amino acidsequence consisting of amino acid residues 21 to 619 of SEQ ID NO: 12,and a protein having an amino acid sequence including substitution,deletion, insertion, and/or addition of one to thirty amino acids in theamino acid sequence consisting of amino acid residues 21 to 619 of SEQID NO: 12, and having activity to selectively link L-phenylalanine to anα-ester site of the L-aspartic acid-α,β-diester through a peptide bond.5. The method for producing an α-L-aspartyl-L-phenylalanine-β-esteraccording to claim 2, wherein the microbe is a transformed microbe thatis capable of expressing a protein selected from the group consistingof: a protein having the amino acid sequence of SEQ ID NO: 12, and aprotein containing a mature protein region, having an amino acidsequence including substitution, deletion, insertion, and/or addition ofone to thirty amino acids in the amino acid sequence of SEQ ID NO: 12,and having activity to selectively link L-phenylalanine to an α-estersite of the L-aspartic acid-α,β-diester through a peptide bond.
 6. Themethod for producing an α-L-aspartyl-L-phenylalanine-β-ester accordingto claim 1, wherein the enzyme is at least one selected from the groupconsisting of: a protein encoded by a nucleotide sequence consisting ofnucleotides 121 to 1917 of SEQ ID NO: 11, and a protein encoded by anucleotide sequence consisting of nucleotides 61 to 1917 of SEQ ID NO:11.
 7. A method of producing an α-L-aspartyl-L-phenylalanine-α-methylester, comprising: synthesizing an α-L-aspartyl-L-phenylalanine-β-methylester by producing an α-L-aspartyl-L-phenylalanine-β-ester according tothe method of claim 1; and converting theα-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.
 8. A method of producing anα-L-aspartyl-L-phenylalanine-α-methyl ester, comprising: synthesizing anα-L-aspartyl-L-phenylalanine-β-methyl ester by producing anα-L-aspartyl-L-phenylalanine-β-ester according to the method of claim 2;and converting the α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.
 9. A method of producing anα-L-aspartyl-L-phenylalanine-α-methyl ester, comprising: synthesizing anα-L-aspartyl-L-phenylalanine-β-methyl ester by producing anα-L-aspartyl-L-phenylalanine-β-ester according to the method of claim 3;and converting the α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.
 10. A method of producingan α-L-aspartyl-L-phenylalanine-α-methyl ester, comprising: synthesizingan α-L-aspartyl-L-phenylalanine-β-methyl ester by producing anα-L-aspartyl-L-phenylalanine-β-ester according to the method of claim 4;and converting the α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.
 11. A method of producingan α-L-aspartyl-L-phenylalanine-α-methyl ester, comprising: synthesizingan α-L-aspartyl-L-phenylalanine-β-methyl ester by producing anα-L-aspartyl-L-phenylalanine-β-ester according to the method of claim 5;and converting the α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.
 12. A method of producingan α-L-aspartyl-L-phenylalanine-α-methyl ester, comprising: synthesizingan α-L-aspartyl-L-phenylalanine-β-methyl ester by producing anα-L-aspartyl-L-phenylalanine-β-ester according to the method of claim 6;and converting the α-L-aspartyl-L-phenylalanine-β-methyl ester toα-L-aspartyl-L-phenylalanine-α-methyl ester.